CN102864119A - Carrier for cell culturing and preparation method of carrier - Google Patents
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Abstract
The invention discloses a carrier for cell culturing. The carrier is a three-dimensional porous graphene support which is fixed on a transparent polystyrene or glass material substrate, liquid tank walls are fixed on the periphery of the three-dimensional porous graphene support to form a culturing tank used for cell culturing. The invention further discloses a preparation method of the carrier for cell culturing. Compared with the prior art, the method includes adopting the three-dimensional porous graphene support infiltrated by solvent before removing a metal catalyst formwork, and removing the air absorbed on the surface of the support by means of vacuum air suction, so that floating of materials on the surface of liquid due to surface tension and density is avoided, and residues of metal and ions can be removed more easily in the process of subsequent treatment. The three-dimensional porous graphene support is used as culturing carrier, realizes three-dimensional culturing of cells, simulates in-vivo growth environment of the cells, is beneficial to maintaining growth states and activity of the cells and accelerates cell growth.
Description
Technical field
The present invention relates to a kind of carrier for cell cultures and preparation method thereof, belong to technical field of biological material.
Background technology
Graphene is a kind of novel carbon nanomaterial, has unique two-dimension plane structure, has been applied to the fields such as flexible and transparent electrode, strongthener; At biomedical sector, Graphene also receives much concern in recent years, has been used to cell imaging, drug delivery, bioanalysis, Stem Cell Engineering and the research of oncotherapy aspect simultaneously.
Spongy three-dimensional porous grapheme material is at first by discoveries such as the Cheng Huiming of Shenyang metal institute, its synthetic method is generally, take nickel foam as catalyzer, organic gas such as methane are carbon source, adopt chemical Vapor deposition process (CVD) preparation to contain the three-dimensional porous grapheme material of catalyzer, then obtain spongy three-dimensional porous grapheme material behind the erosion removal catalyzer.The grapheme material of this method preparation has good electricity, mechanical property, can further be applied to the biomedical sectors such as biochip, embedded material.
Cell in the histoorgan is all grown in three-dimensional environment, therefore on the Tissue Culture Dish of present two dimensional surface or graphene substrate during culturing cell, be subjected to the planar growth environmental limit, compared very large change under the activity of cell, pattern and growth conditions etc. and the internal milieu, therefore explored and spongy three-dimensional porous Graphene is applied to cell cultures has important value; And, because cell cultures has very high requirement to foreign matter content and the course of processing of material, how to prepare have high conductivity, the three-dimensional porous Graphene of ultralight porous, good biocompatibility also is a difficult problem that needs to be resolved hurrily.
Summary of the invention
Problem for the above-mentioned prior art existence of mentioning, the invention provides a kind of carrier for cell cultures and preparation method thereof, this cell culture vector has been realized the dimensional culture of cell, the tumor growth environment of analog cell, be conducive to keep growth conditions and the activity of cell, Promote cell's growth.
For achieving the above object, the present invention has adopted following technical scheme:
A kind of carrier for cell cultures, described carrier are three-dimensional porous Graphene support.
Further, the appearance of described three-dimensional porous Graphene support is modified with bioactive molecules, and described bioactive molecules can be poly-lysine or ln.
Preferably, the aperture of described three-dimensional porous Graphene support is the 20-500 micron, and density is 2-20 mg/cm
3
Described three-dimensional porous Graphene support is fixed in transparent polystyrene or the glass material substrate, and around three-dimensional porous Graphene support the stationary liquid pool wall, form cultivation pool, be used for cell cultures; Institute's cultured cells be at least tumour cell, former generation neurocyte and neural stem cell in one or more.
The preparation method of aforesaid carrier for cell cultures, it may further comprise the steps:
(a) the applied chemistry gas-phase deposition is at the three-dimensional porous Graphene support of metal foam support preparation;
The three-dimensional porous Graphene that (b) will contain the metal foam support is immersed in 5-20 min in the mixed solvent of alcohol and water; Adopt vacuum suction to remove the air of three-dimensional porous Graphene rack surface absorption;
(c) in closed container, adopt with acid etching solution and remove the metal foam support, then adopt successively the hydrochloric acid of 1,0.1,0.01,0.001 mol/L or salpeter solution respectively to wash 1 time, again with deionized water wash for several times; Obtain the carrier for cell cultures: three-dimensional porous Graphene support.
Preferably, in the step (b) behind the vacuum suction pressure and kept at least 30 minutes less than 10kPa.
Preferably, described metal catalyst is nickel foam, foam copper, foam gold copper or nickel foam copper alloy.
Compared with prior art, advantage of the present invention is at least:
(1) cell culture vector provided by the invention, culturing cell on three-dimensional porous Graphene supporting structure, because the vesicular structure of three-dimensional porous grapheme material has the superelevation porosity, for Growth of Cells provides adequate space, realize the dimensional culture of cell, simulate the tumor growth environment of cell, be conducive to keep growth conditions and the activity of cell, Promote cell's growth; And can keep the good mechanics of intrinsic Graphene, electricity, chemical property through the three-dimensional porous Graphene that present method is processed, satisfy the demands such as regulating cell growth and differentiation;
(2) the present invention is in the process of preparation as the three-dimensional porous Graphene support of substrate, before removing the catalyzer template, at first adopt solvent to infiltrate three-dimensional porous Graphene support, avoid material to swim in fluid surface because of the reason of surface tension and density, and vacuumize the air of removing material surface absorption, improve the wetting property of corrosive fluid, and in enclosed environment, carry out immersion treatment, effectively avoided the volatilization of corrosive fluid and condensed, the easier remnants that in subsequent processes, remove metal and ion, thus significantly improve the biocompatibility of material and the repeatability for the treatment of processes; Effectively avoid catalyzer and etching reagent to remain in the Graphene support, improved activity and the survival rate of culturing cell.
Description of drawings
In order to be illustrated more clearly in the embodiment of the present application or technical scheme of the prior art, the below will do to introduce simply to the accompanying drawing of required use in embodiment or the description of the Prior Art, apparently, the accompanying drawing that the following describes only is some embodiment that put down in writing among the application, for those of ordinary skills, under the prerequisite of not paying creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is the structural representation that contains the cultivation pool of three-dimensional porous Graphene support in a preferred embodiment of the present invention;
Fig. 2 is that the three-dimensional porous Graphene rack surface in the embodiment of the invention is cultivated PC 12 cells, the fluorescence photo behind 'beta '-tubulin (Tubulin) immunofluorescence dyeing;
Fig. 3 is the stereoscan photograph of three-dimensional porous Graphene support in the embodiment of the invention.
Embodiment
For shortcomings and deficiencies of the prior art, utilize three-dimensional porous Graphene support as the preparation method of the conception of cell culture vector and this carrier for cell cultures through studying for a long period of time and put into practice, the present invention proposes.
The carrier that should be used for cell cultures is three-dimensional porous Graphene support 2; The appearance of described three-dimensional porous Graphene support 2 can be modified with bioactive molecules, and described bioactive molecules can be poly-lysine or ln; The aperture of described three-dimensional porous Graphene support 2 is the 20-500 micron, and density is 2-20 mg/cm
3
As shown in Figure 1, described three-dimensional porous Graphene support 2 is fixed in transparent polystyrene or the glass material substrate 1, and around three-dimensional porous Graphene support stationary liquid pool wall 4, form cultivation pool; Then adopt acetone, Virahol, alcohol wash to remove organic substance residues, soak three-dimensional porous Graphene support 2 with further removal solubility toxic substance with a large amount of deionized waters again; In cultivation pool, inject substratum and form liquid pool 3, be used for cell cultures; Institute's cultured cells be at least tumour cell, former generation neurocyte and neural stem cell in one or more.
The present invention also provides a kind of making to be used for the preparation method of the carrier of cell cultures, it mainly comprises the following steps: at first, adopt the method for traditional C VD, be about 95% metal foam take porosity and be catalyzer, under 1000 ℃ of conditions, prepare individual layer or few layer graphene take methane as carbon source, be prepared with the three-dimensional porous Graphene support of metal foam; Secondly, the three-dimensional porous Graphene that will contain metal foam is immersed in 5-20 min in the mixed solvent of alcohol and water, adopts vacuum suction to remove the air of three-dimensional porous Graphene rack surface absorption; Then, in closed container, adopt and remove metal foam with acid etching solution, adopt successively at last the hydrochloric acid of 1,0.1,0.01,0.001 mol/L or salpeter solution respectively to wash once, obtain for several times three-dimensional porous Graphene support with deionized water wash again.Wherein, the aperture of three-dimensional porous Graphene support determines that by the aperture structure of nickel foam density is relevant with the number of plies of material.Preferably, the three-dimensional porous Graphene support number of plies is five to ten layers, and the aperture is between the 100-300 micron, and density is 2-20 mg/cm in density
3
By the present invention, can realize simultaneously various types of cells in the dimensional culture on Graphene surface, for further graphene-based biomedical applications provides technical support.
Below in conjunction with some preferred embodiments technical scheme of the present invention is described further.
Adopt the method for CVD, take porosity 95 %, aperture in the nickel foam of 30-300 micron as catalyzer, under 900 ℃ of conditions, take methane as carbon source for growth 5-10 layer graphene.The three-dimensional porous Graphene support that will contain catalyzer nickel is immersed in 10 min in the aqueous solution that contains 75 % alcohol, and bleeding makes pressure less than 10 kPa, keeps 30 min, removes the air of three-dimensional porous Graphene rack surface absorption; Then pass into air to normal pressure, add the acidic ferric chloride corrosive fluid of 1000 times of volumes, in sealed environment, soak 24 h to remove catalyzer nickel; Then adopt successively the hydrochloric acid soln of 1,0.1,0.01,0.001 mol/L respectively to wash 1 time, use again deionized water wash 3 times.The dry back aperture of the three-dimensional porous Graphene support that makes slightly reduces than the aperture of nickel foam template between the 20-300 micron, and density is about 4-5 mg/cm
3
With substrate of glass three-dimensional porous Graphene support is picked up, directly adopts DOW CORNING 3140 silicone adhesive agents to fix, with the liquid pool wall be fixed in three-dimensional porous Graphene support around, and at ambient temperature set times 12 h, form cultivation pool; Then use acetone treatment 5 h, clean 0.5 h with Virahol subsequently, then use a large amount of washed with de-ionized water, and soak 120 h.The carrier that is used for cell cultures that makes is soaked in poly-lysine PBS solution 12 h of 1 mg/mL, then washes with a large amount of sterilization PBS damping fluids.
Inject in the cultivation pool and contain 15% blood serum medium, and soak 2 h, be used for the cultivation of PC12 cell (the adult rat adrenal tissue medullary substance is had a liking for the strain of chromium knurl noble cells), substratum is the DMEM that contains 15 % foetal calf serums, and inoculum density is 10
5Cell/cm
2Cultivation results shows that the cell cell space of growth is plentiful, endochylema is transparent, the cell edges refractive index is high, and growth conditions is good, as shown in Figure 2.
Adopt the method for CVD, take porosity 95 %, aperture at the foam copper of 100-300 micron as catalyzer, under 950 ℃ of conditions, take methane as carbon source for growth 10-20 layer graphene.The three-dimensional porous Graphene support that will contain catalyzer nickel is immersed in 20 min in the aqueous solution that contains 98 % alcohol, and bleeding makes pressure to 100 Pa, keeps 30 min, removes the air of three-dimensional porous Graphene rack surface absorption; Then pass into air to normal pressure, add the acid etching solution of 100 times of volumes to remove catalyzer nickel; Then adopt successively the salpeter solution of 1,0.1,0.01 mol/L respectively to wash 1 time, use again deionized water wash 6 times.The dry back aperture of the three-dimensional porous Graphene support that makes is between the 100-300 micron, and density is about 5-8 mg/cm
3, pattern as shown in Figure 3.
With substrate of glass three-dimensional porous graphite support alkene is picked up, directly adopts DOW CORNING 3140 silicone adhesive agents to fix after dry, with the liquid pool wall be fixed in three-dimensional porous Graphene support around, and at ambient temperature set time 24h, form cultivation pool; Then use acetone treatment 2 h, use subsequently alcohol wash 2 h, then use a large amount of washed with de-ionized water, and soak 24 h.The carrier that is used for cell cultures that makes is soaked in poly-lysine PBS solution 12 h of 1 mg/mL, then washes with a large amount of sterilization PBS damping fluids; In cultivation pool, inject the DMEM substratum that contains 15% serum, can be used for the PC12 cell cultures.
Adopt the method for CVD, take porosity 95 %, aperture at the foam gold copper of 300-500 micron as catalyzer, under 750 ℃ of conditions, take methane as carbon source for growth 50-100 layer graphene.The three-dimensional porous Graphene support that will contain catalyzer nickel is immersed in 60 min in the aqueous solution that contains 2 % alcohol, and bleeding makes pressure less than 10 kPa, keeps 30 min, removes the air of three-dimensional porous Graphene rack surface absorption; Then pass into air to normal pressure, add the acidic ferric chloride corrosive fluid of 1000 times of volumes to remove catalyzer nickel; Then adopt successively 1,0.1,0.01,0.001, the hydrochloric acid soln of 0.0001 mol/L respectively washs 1 time, uses deionized water wash 3 times again.The dry back aperture of the three-dimensional porous Graphene support that makes is between the 300-500 micron, and density is about 15-20 mg/cm
3
Polystyrene (TCPS) substrate that using-system is cultivated purposes picks up three-dimensional porous Graphene, directly adopt DOW CORNING 3140 silicone adhesive agents to fix, with the liquid pool wall be fixed in three-dimensional porous Graphene support around, and at ambient temperature set time 48h, form cultivation pool; Then process 5 h with alcohol, then use a large amount of washed with de-ionized water, and soak 1 h.The carrier that is used for cell cultures that makes is soaked in poly-lysine PBS solution 24 h of 10 mg/mL, then washes with a large amount of sterilization PBS damping fluids.
Inject in the cultivation pool and contain 15% blood serum medium, and soak 2 h, be used for the cultivation of former generation hippocampal neurons, substratum is the DMEM that contains 15% foetal calf serum, and inoculum density is 10
6Cell/cm
2Cultivation results shows that the biocompatibility of three-dimensional porous Graphene support is good, and cell growth state, MTT detected result show that cell growth state all is better than its growth conditions on two dimensional surface Graphene and TCPS substrate, and cell survival rate improves 50 %.
Adopt the method for CVD, take porosity 95 %, aperture at the foam copper nickelalloy of 100-300 micron as catalyzer, under 900 ℃ of conditions, take methane as carbon source for growth 1-5 layer graphene.The three-dimensional porous Graphene support that will contain catalyzer nickel is immersed in 5 min in the aqueous solution that contains 50 % alcohol, and bleeding makes pressure less than 100 Pa, keeps 30 min, removes the air of three-dimensional porous Graphene rack surface absorption; Then pass into air to normal pressure, add the acid iron nitrate corrosive fluid of 100 times of volumes to remove catalyzer nickel; Then adopt successively the salpeter solution of 0.1,0.01 mol/L respectively to wash 1 time, use again deionized water wash 3 times.The dry back aperture of the three-dimensional porous Graphene support that makes slightly reduces than the aperture of nickel foam template between the 50-300 micron, and density is about 2-3 mg/cm
3
With the TCPS substrate three-dimensional porous Graphene support is picked up, directly adopts DOW CORNING 3140 silicone adhesive agents to fix, with the liquid pool wall be fixed in three-dimensional porous Graphene support around, and at ambient temperature set time 24h, form cultivation pool; Then use acetone treatment 5 h, then use a large amount of washed with de-ionized water, and soak 48 h.The carrier that is used for cell cultures that makes is soaked in poly-lysine PBS solution 12 h of 1 mg/mL, then washes with a large amount of sterilization PBS damping fluids.Inject in the cultivation pool and contain 15% blood serum medium, and soak 10 h, be used for the cultivation of former generation hippocampal neurons.
Embodiment 5
Adopt the method for CVD, take porosity 95 %, aperture in the nickel foam of 100-300 micron as catalyzer, under 900 ℃ of conditions, take methane as carbon source for growth 5-10 layer graphene.The three-dimensional porous Graphene support that will contain catalyzer nickel is immersed in 20 min in the aqueous solution that contains 75 % alcohol, and bleeding makes pressure less than 10 kPa, keeps 50 min, removes the air of three-dimensional porous Graphene rack surface absorption; Then pass into air to normal pressure, add the acidic ferric chloride corrosive fluid of 1000 times of volumes, in sealed vessel, soak 24 h to remove catalyzer nickel; Then adopt successively the hydrochloric acid soln of 1,0.1,0.01,0.001 mol/L respectively to wash 1 time, use again deionized water wash 6 times.The dry back aperture of the three-dimensional porous Graphene support that makes slightly reduces than the aperture of nickel foam template between the 100-300 micron, and density is about 4-5 mg/cm
3
With substrate of glass three-dimensional porous Graphene is picked up, directly adopts DOW CORNING 3140 silicone adhesive agents to fix, with the liquid pool wall be fixed in three-dimensional porous Graphene support around, and at ambient temperature set times 12 h, form cultivation pool; Then respectively process 1 h with acetone, Virahol and alcohol, then use a large amount of washed with de-ionized water, and soak 120 h.Adopt biocompatible molecular layer Fibronectin 0.1 μ g/mL to modify 120 h on the three-dimensional porous Graphene support in the cultivation pool, then with a large amount of sterilization PBS damping fluid flushings.
Adopt the Screening of Media method of selecting, cultivate and obtain neural ball sample mouse neural stem cell, adopt the method for unicellular dispersion at three-dimensional porous Graphene rack surface inoculating cell, density is 10
4Cell/cm
2MTT and LDH enzymic activity detected result show two days later, this three-dimensional porous Graphene carrier has good biocompatibility, with the TCPS(control group) compare, survival rate is 98% of control group absorbance value, the LDH analytical results is 101% of control group fluorescent intensity, and the relative cell survival rate of surface both, film integrality are without statistically-significant difference; And before removing the catalyzer template, do not adopt solvent to infiltrate three-dimensional porous Graphene support, and be～400% for～40%, LDH enzymic activity (being inversely proportional to cell membrane integrity) only without vacuumizing the standby three-dimensional grapheme substrate surface cell survival rate of air-making of removing material surface absorption.
Embodiment 6
Adopt the method for CVD, take porosity 90 %, aperture in the nickel foam of 100-400 micron as catalyzer, under 900 ℃ of conditions, take methane as carbon source for growth 10-20 layer graphene.The three-dimensional porous Graphene that will contain catalyzer nickel is immersed in 20 min in the aqueous solution that contains 75 % alcohol, and bleeding makes pressure less than 100 Pa, keeps 30 min, removes the air of three-dimensional porous Graphene rack surface absorption; Then pass into air to normal pressure, add acidic ferric chloride (1 mol/L) solution of 1000 times of volumes, corrosion 120 h remove catalyzer nickel in closed environment; Then adopt successively the hydrochloric acid soln of 1,0.1,0.01,0.001 mol/L respectively to wash 1 time, use again deionized water wash 6 times.The dry back aperture of the three-dimensional porous Graphene support that makes is between the 50-300 micron, and density is about 4-6 mg/cm
3
With substrate of glass three-dimensional porous Graphene is picked up, directly adopts DOW CORNING 3140 silicone adhesive agents to fix, with the liquid pool wall be fixed in three-dimensional porous Graphene support around, and at ambient temperature set times 12 h, form cultivation pool; Then respectively process 2 h with acetone, Virahol and alcohol, then use a large amount of washed with de-ionized water, and soak 120 h.Inject in the cultivation pool and contain the DMEM substratum of 10 % serum, and soak 12 h, be used for the cultivation of Marrow Mesenchymal Stem Cells.
Need to prove, in this article, term " comprises ", " comprising " or its any other variant are intended to contain comprising of nonexcludability, thereby not only comprise those key elements so that comprise process, method, article or the equipment of a series of key elements, but also comprise other key elements of clearly not listing, or also be included as the intrinsic key element of this process, method, article or equipment.Do not having in the situation of more restrictions, the key element that is limited by statement " comprising ... ", and be not precluded within process, method, article or the equipment that comprises described key element and also have other identical element.
The above only is the application's embodiment; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the application's principle; can also make some improvements and modifications, these improvements and modifications also should be considered as the application's protection domain.
Claims (8)
1. a carrier that is used for cell cultures is characterized in that described carrier is three-dimensional porous Graphene support.
2. the carrier for cell cultures according to claim 1 is characterized in that, the appearance of described three-dimensional porous Graphene support is modified with bioactive molecules.
3. the carrier for cell cultures according to claim 2 is characterized in that, described bioactive molecules is poly-lysine or ln.
4. the carrier for cell cultures according to claim 1 and 2 is characterized in that, the aperture of described three-dimensional porous Graphene support is the 20-500 micron, and density is 2-20 mg/cm
3
5. the carrier for cell cultures according to claim 1 is characterized in that, this carrier institute cultured cells be at least tumour cell, former generation neurocyte and neural stem cell in one or more.
6. the preparation method of the arbitrary described carrier for cell cultures of claim 1 to 5, its feature
Be that it may further comprise the steps:
(a) preparation contains the three-dimensional porous Graphene support of metal foam;
(b) mixed solvent with alcohol and water infiltrates the three-dimensional porous Graphene support that step (a) obtains, and adopts vacuum suction to remove the air of three-dimensional porous Graphene rack surface absorption;
(c) in closed container, remove the metal foam in the three-dimensional porous Graphene support.
7. the carrier preparation method for cell cultures according to claim 6 is characterized in that, described metal foam is nickel foam, foam copper, foam gold copper or nickel foam copper alloy.
8. the carrier preparation method for cell cultures according to claim 6 is characterized in that, in the step (b) behind the vacuum suction pressure and kept at least 30 minutes less than 10kPa.
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| CN104531618A (en) * | 2014-12-21 | 2015-04-22 | 朱熹 | Method for obtaining lots of three-dimensional growing tumour cells at low cost by utilizing graphene nano material |
| CN106701656A (en) * | 2015-07-27 | 2017-05-24 | 天津卫凯生物工程有限公司 | Composite scaffold for cell culture and preparation method thereof |
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| WO2017028714A1 (en) * | 2015-08-18 | 2017-02-23 | 重庆润泽医药有限公司 | Culture device for tissue cell |
| CN108300713A (en) * | 2017-12-31 | 2018-07-20 | 宁波大学 | The method and device of fixed cell |
| CN109593704A (en) * | 2019-01-31 | 2019-04-09 | 北京华龛生物科技有限公司 | A kind of method of three-dimensional microcarrier cell absorption culture |
| CN111501350A (en) * | 2020-05-08 | 2020-08-07 | 广东工业大学 | Biological scaffold for inducing growth of nerve cells in vitro and preparation method and application thereof |
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| CN114134196A (en) * | 2021-12-07 | 2022-03-04 | 浙江大学 | Method and device for simulating effect of toxin on neural stem cells by crossing blood brain barrier |
| CN114134196B (en) * | 2021-12-07 | 2024-06-28 | 浙江大学 | Method and device for simulating toxin to cross blood brain barrier to act on neural stem cells |
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