CN102559494B - Cell culture device with surface coating - Google Patents
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Abstract
The invention discloses a cell culture device with a surface coating. The cell culture device with the surface coating comprises a plastic base layer and the surface coating, wherein the surface coating is a nanomaterial layer; the contact angle of the surface coating and water is more than 10 degrees; the fractal dimension of the surface coating is 2.2-2.3; and the nanomaterial layer is laid or coated on the plastic base layer. The invention further discloses a cell culture method. In the cell culture method, cells are planted on the cell culture device with the surface coating and cultured,so that the cells can keep shapes and functions closer to those in a body. The cell culture device with the surface coating is convenient to manufacture, good in stability and good in biocompatibility, can facilitate the cell growth, can make contribution to understanding functional characteristics of the cells in an in-vivo physiological state and evaluating functions of the cells in a pathological state, and has high practicality and applicability.
Description
Technical field
The present invention relates to a kind of cell culture apparatus with top coat, also relate to a kind of method for cell cultures.
Background technology
The external environment of cell growth has significant effects to growthhabit and the function of cell.In vivo, cell is surrounded by cell exocoel like the complicated scaffolding, and it affects the motion of polarization, growth and the cell of cell.So, in cellular system engineering or experiment in vitro, aseptic except need, keep proper temperature and provide that physiological environment makes cells survival, the growth in certain nutritional condition analogue body, the external environment that also is necessary very much to make up a kind of cell cultures makes cell can keep a kind of more near the form in the body, is conducive to understand cell functional character and the effect of assessment cell under pathological state under the physiological status in vivo.
Common vitro culture is used polystyrene synthetic culturing bottle ware surface or other general surface, these surfaces do not have hydrophobicity to water or substratum, contact angle is about 30 degree, it is a two-dimentional culture environment, the restriction that cell is subjected on this surface is less relatively and usually be the form of tiling, and functional character also changes.All the time, the investigator is seeking a kind ofly to make the vitro culture mode of keeping the cell natural form, obtained certain development at present, wherein use the cultivation material of three dimensional form to carry out the focus that dimensional culture is research, for example: patent CN101484574A discloses a kind of cell cultures substrate that contains the high internal phase ratio emulsions polymkeric substance of polymerization, being used for conventional cell three-dimensional after adjusting and modifying cultivates, the typical mammalian cell that is used for is cultivated, and also discloses the propagation of the using method of this substrate in cell culture system with the research and analysis cell, differentiation and functionalization; Patent CN1973029A discloses a kind of nanofibrils structure that is used for cell cultures and organizational project, in cell cultures, be mainly used in the environment of cell proliferation and/or differentiation, wherein the nanofibrils structure is limited by one or more nanofiber networks, and nanofiber comprises that diameter is about 1000 nanometers or polymeric fine fibers still less; Also have the investigator to use the function of studying cell such as the method for tissue slice or organ culture.Though these methods are conducive to analog cell environment in vivo, but still there is some deficiency: bother as its preparation of substrate that is used for cell cultures, wayward; Same substrate is used for same cell cultures, may produce different results, and is namely repeated bad; Cell survival rate may be affected during cultivation; For the structure function of the single kind of research cell, target cell is grown in and causes observing relatively difficulty in the culture medium especially, and training method is relatively complicated, so be not widely used in the research of single kind or individual cells.
Summary of the invention
The object of the present invention is to provide a kind of easy to prepare, good stability, good biocompatibility to be easy to the cell growth, be conducive to make up the cell culture apparatus with top coat that is similar to the extracellular environment in the body, it can prevent adhesions such as dust, grease, microbial spore, dirt, and making cell can keep a kind of more near the form in the body, the functional character under the physiological status and assessment cell act under pathological state in vivo thereby be conducive to understand cell; Another object of the present invention is to provide a kind of method for cell cultures.
Above-mentioned technical purpose of the present invention is achieved by the following technical programs: a kind of cell culture apparatus with top coat, it comprises plastic basic unit and top coat, described top coat is layer of nanomaterial, and top coat is 2.2 ~ 2.3 to the contact angle of water greater than 150 degree, fractal dimension.
Above-mentioned contact angle has super-hydrophobicity greater than the nano material of 150 degree, can effectively prevent the adhesion of dust, grease, dirt etc., fractal dimension has reflected the spacial validity of complicated form, experimental studies have found that fractal dimension is 2.2 ~ 2.3, contact angle can make up the extracellular environment that is similar in the body greater than the nano materials of 150 degree, be conducive to cell and keep the interior morphological structure of analog, have better biocompatibility and be easy to the object observing cell.
In order to obtain more excellent security and sterility, as of the present invention preferred, described layer of nanomaterial is sprawled or is coated on the plastic basic unit.
As of the present invention preferred, above-mentioned cell culture apparatus with top coat can obtain as follows:
(1) with tripalmitin 65 ~ 70
o Melt 10 ~ 15 min under the C;
(2) sprawl or be coated on the tripalmitin of fusion on the plastic basic unit;
(3) quick cooled and solidified post-heating to 40 ~ 45 in 10 ~ 20 min
oInsulation 10 hr behind the C, the surperficial contact angle to water of formation are 2.23 ± 0.03 the cell culture apparatus with top coat greater than 150 degree, fractal dimension.
As of the present invention preferred, the too thin then top coat of the thickness of sprawling or applying in the described step (2) easily peels off, and is too thick, is unfavorable for cell observation, selects 50 ~ 100 μ m comparatively suitable.
Another is preferred as of the present invention, and above-mentioned cell culture apparatus with top coat can also obtain as follows:
(1) with alkyl ketene dimer 70 ~ 75
oMelt 5 ~ 10 min under the C;
(2) sprawl or be coated on the alkyl ketene dimer of fusion on the plastic basic unit;
(3) quick cooled and solidified post-heating to 45 ~ 50 in 10 ~ 15 min
oKeep 40 min behind the C, the surperficial contact angle to water of formation is 2.27 ± 0.03 the cell culture apparatus with top coat greater than 150 degree, fractal dimension.
As of the present invention preferred, the too thin then top coat of the thickness of sprawling or applying in the described step (2) easily peels off, and is too thick, is unfavorable for cell observation, selects 60 ~ 80 μ m comparatively suitable.
As of the present invention preferred, described plastic basic unit for cell cultures is a kind of of culture dish, culture plate, culturing bottle.
The present invention also is provided for the method for cell cultures, is cell seeding is cultivated on above-mentioned arbitrary described cell culture apparatus with top coat, makes cell can keep a kind of more near form and function in the body.
In sum, the present invention compared with prior art has following outstanding advantage and beneficial effect:
1. top coat of the present invention is the super-hydrophobicity layer of nanomaterial, can reduce the coatingsurface energy, give coatingsurface super-hydrophobic ability, good stability, can effectively prevent adhesions such as dust, grease, microbial spore, dirt, provide a kind of aseptic dustless cells in vitro culture environment thereby be beneficial to;
2. discover, top coat of the present invention can make up the extracellular environment that is similar in the body, it is a kind of more near the morphological structure in the body that cell is kept, thereby be conducive to understand cell functional character and the effect of assessment cell under pathological state under the physiological status in vivo;
3. the present invention is easy to prepare, has better biocompatibility, is easy to the object observing cell; The cell of growing at top coat of the present invention can be used for immunochemistry dyeing, hybridization in situ experiment, cell composition and also can be used for qualitative or quantitative analysis, has very strong practicality and applicability.
Description of drawings
Fig. 1 is surface scan Electronic Speculum (SEM) photo of the cell culture apparatus of embodiment 1: A voltage 5.0KV, Bar=40 μ m; B voltage 10.0KV, Bar=80 μ m.
Fig. 2 is the water photo on the cell culture apparatus of embodiment 1A and embodiment 2B (Bar=500 μ m) respectively.
Fig. 3 is the photo (Bar=25 μ m) of Rat Astroglia after carrying out skelemin actin dyeing behind the different culture apparatuses cultivation different times: A ordinary cells culture apparatus; B is the cell culture apparatus with top coat of embodiment 1.
Comparison (the * of the primary process number that Fig. 4 cultivates at different culture apparatuses for Rat Astroglia, branch's number, projection length ratio, cell fractal dimension
P＜0.05; *
P＜0.01; * *
P＜0.001).
Fig. 5 is Rat Astroglia cultured cells mortality ratio (adopting Hoechst/PI dyeing) (* on different culture apparatuses
P＜0.05).
Fig. 6 is the photo (Bar=50 μ m) of rat glioma C6 cell after carrying out skelemin actin dyeing behind the different culture apparatuses cultivation different times: A ordinary cells culture apparatus; B is the cell culture apparatus of embodiment 1.
Fig. 7 is the surperficial SEM photo of the cell culture apparatus of embodiment 2: A voltage 5.0KV, Bar=100 μ m; B voltage 5.0KV, Bar=80 μ m.
Fig. 8 is the photo (Bar=50 μ m) of astroglia cell after carrying out skelemin actin dyeing behind different culture apparatuses cultivation 2 d: A ordinary cells culture apparatus; B is the cell culture apparatus of embodiment 2.
Fig. 9 carries out skelemin actin dyeing back photo (Bar=20 μ m) for rat glioma C6 cell after different culture apparatuses are cultivated 2 d: A ordinary cells culture apparatus; B is the cell culture apparatus of embodiment 2.
Embodiment
Below in conjunction with embodiment and accompanying drawing the present invention is described in further detail.This specific embodiment only is explanation of the invention; it is not the restriction to invention; those skilled in the art can make any modification to present embodiment as required after reading this specification sheets, but as long as all are subjected to the protection of patent law in claim scope of the present invention.
A kind of cell culture apparatus with top coat, it obtains as follows:
(1) with tripalmitin 65 ~ 70
o Melt 10 ~ 15 min under the C;
(2) tripalmitin of fusion is sprawled or is coated on the plastic culture dish, sprawl or coating thickness is 50 ~ 100 μ m;
(3) quick cooled and solidified post-heating to 40 ~ 45 in 10 ~ 20 min
oInsulation 10 hr behind the C.The SEM photo on this cell culture apparatus surface is seen Fig. 1, wherein Biao Mian cross section (Figure 1B) SEM image is 2.23 ± 0.03 with box-counting method calculating fractal dimension, water is seen Fig. 2 A at its surperficial photo, measuring this surface with contact angle measurement is 150 ~ 160 degree to the contact angle of water, shows that this material has super-hydrophobicity.
The neurogliocyte that is star in the vertebrates central nervous system is astroglia cell, the outer ion of its adjustable ganglion cell and chemical environment, support brain blood barrier, for nervous tissue provides nutritive substance, it is the class cell the most widely that distributes in the mammal brain, also be a kind of of volume maximum in the spongiocyte, have the meticulous three-dimensional arrangement of a large amount of projections in vivo.But when ordinary cells culture apparatus such as plastics or the cultivation of glass culture surface, astroglia cell often is flat multiangular, loses a large amount of projections.
We plant the cortex astroglia cell of rat and cultivate on above-mentioned cell culture apparatus with top coat that (substratum is that DMEM contains 10% foetal calf serum, 37
oC, 5% CO
2), cultivate 1 d, 2 d, (dyestuff is rhodamine-phalloidin to carry out cytoskeleton actin dyeing behind 3 d, MolecularProbes), under fluorescent microscope, take pictures then and see Fig. 3 B, cultivate the identical time to planting in the astroglia cell on ordinary cells culture apparatus surface under the same conditions, carry out cytoskeleton actin dyeing then, photo is seen Fig. 3 A, contrast A can find with B: the astroglia cell appearance is significantly different, be polygonal flat shape on ordinary cells culture apparatus surface, demonstrate heterogeneous and inorganization, and astroglia cell becomes many projections state at tripalmitin super-hydrophobic nano material, have complicated morphological structure, more approaching with form in the body.
We have also compared at astrocytes cultured on the ordinary cells culture apparatus and projection number (Fig. 4 A with astrocytes cultured on the cell culture apparatus of top coat, B), fractal dimension (Fig. 4 D of projection length (Fig. 4 C) and cell, the index of reflection cellularstructure complicacy), discovery the primary process number of astroglia cell on the cell culture apparatus with tripalmitin super-hydrophobic nano material surface coating and branch's number (comprising secondary and above projection number thereof) obviously more than common material on astroglia cell, projection length ((cell centre is to the difference of the interior radius of circle of projection top length-cell) * 100%/cell centre is to projection top length) obviously is longer than astroglia cell on the ordinary cells culture apparatus relatively, and while cell fractal dimension is obvious fractal dimension greater than the astroglia cell on the ordinary cells culture apparatus also.Show thus, on tripalmitin super-hydrophobic nano material, the astroglia cell form more is conducive to understand the functional character under the physiological status and the effect of assessment cell under pathological state in the cell paste more near the long complicated form of the many projections in the body, projection.
We have also further compared the mortality ratio (Fig. 5) of astrocytes cultured on the cell culture apparatus with tripalmitin super-hydrophobic nano material surface coating and on the ordinary cells culture apparatus, the cultured cells mortality ratio is lower than the mortality ratio of cultivating at the ordinary cells culture apparatus on the cell culture apparatus of top coat having in discovery, mortality ratio when particularly cultivating 2 d is starkly lower than the astroglia cell mortality ratio on the ordinary cells culture apparatus, shows that the cell culture apparatus with top coat has better biocompatibility.
In addition, also rat glioma C6 cell (can win from Shanghai credit bio tech ltd buy) is planted and cultivate in above-mentioned cell culture apparatus with top coat that (DMEM contains 10% foetal calf serum, 37
oC, 5% CO
2), cultivate and carry out cytoskeleton actin dyeing behind 2 d, under fluorescent microscope, take pictures then (Fig. 6 B).Under the same conditions to planting the same identical time of glioma C6 cell cultures in ordinary cells culture apparatus surface, carry out cytoskeleton actin dyeing then, photo is seen Fig. 6 A, contrast A and B can find: rat glioma cell C6 is bipolar form on common thin culture apparatus surface, and become the multiple-limb state at tripalmitin super-hydrophobic nano material, a large amount of tiny projections is arranged, show that its structure is more complicated, point out this material can make cell keep a kind of more near the form in the body.
A kind of cell culture apparatus with top coat, it obtains as follows:
(1) with alkyl ketene dimer 70 ~ 75
oMelt 5 ~ 10 min under the C;
(2) alkyl ketene dimer of fusion is sprawled or is coated on plastic culture plate or the culturing bottle, the thickness of sprawling or applying is 60 ~ 80 μ m;
(3) quick cooled and solidified post-heating to 45 ~ 50 in 10 ~ 15 min
oKeep 40 min behind the C.The SEM photo on this cell culture apparatus surface is seen Fig. 7.Wherein Biao Mian cross section (Fig. 7 B) SEM image is used for the box-counting method to calculate fractal dimension is 2.27 ± 0.03, and measuring this surface with contact angle measurement is 150 ~ 160 degree (Fig. 2 B) to the contact angle of water, shows that this surface has super-hydrophobicity.
The rat layer astroglia cell planted on the cell culture apparatus with alkyl ketene dimer super-hydrophobic nano material cultivate that (substratum is that DMEM contains 10% foetal calf serum, 37
oC, 5% CO
2), cultivate and carry out cytoskeleton actin dyeing after 3 days, under fluorescent microscope, take pictures then and see Fig. 8 B; Cultivate the identical time to planting in the astroglia cell on ordinary cells culture apparatus surface under the same conditions, cytoskeleton actin dyeing photo after the cultivation is seen Fig. 8 A, contrast A can find with B: the astroglia cell appearance is significantly different, be flat polygonal form on ordinary cells culture apparatus surface, and on the cell culture apparatus surface with alkyl ketene dimer super-hydrophobic nano material surface coating more projection is arranged, more approaching with form in the body.
In addition, also rat glioma cell C6 kind is planted and cultivate on above-mentioned cell culture apparatus with top coat that (DMEM contains 0.1% foetal calf serum, 1mM dbcAMP, 37
oC, 5% CO
2), carry out cytoskeleton actin dyeing after cultivating 2 d, Fig. 9 B then takes pictures under fluorescent microscope, cultivate the identical time to planting in the same glioma cell C6 on ordinary cells culture apparatus surface under the same conditions, cytoskeleton actin dyeing photo after the cultivation is seen Fig. 9 A, contrast A and B can find: rat glioma C6 cell is polygonal flat shape on ordinary cells culture apparatus surface, and have the cell culture apparatus surface one-tenth multiple-limb state of top coat, a large amount of tiny projections is arranged, and three-dimensional structure is observed into solid shape.Find behind the analysis dimension of analysis of cells that the fractal dimension of rat glioma C6 cell on alkyl ketene dimer super-hydrophobic nano material is 1.27 ± 0.06, and general surface is 1.14 ± 0.04(
P＜0.05), shows that its structure is more complicated, point out this material can make cell keep a kind of more near the form in the body.
Not detailed part is technology well-known to those skilled in the art among the present invention.
Claims (4)
1. cell culture apparatus with top coat, it is characterized in that: it comprises plastic basic unit and top coat, described top coat is layer of nanomaterial; Described layer of nanomaterial is sprawled or is coated on the plastic basic unit; It obtains as follows:
(1) with tripalmitin 65 ~ 70
oMelt 10 ~ 15 min under the C;
(2) sprawl or be coated on the tripalmitin of fusion on the plastic basic unit;
(3) quick cooled and solidified post-heating to 40 ~ 45 in 10 ~ 20min
oInsulation 10 hr behind the C, the surperficial contact angle to water of formation are 2.23 ± 0.03 the cell culture apparatus with top coat greater than 150 degree, fractal dimension.
2. according to the described a kind of cell culture apparatus with top coat of claim 1, it is characterized in that: sprawl in the described step (2) or coating thickness is 50 ~ 100 μ m.
3. according to the arbitrary described a kind of cell culture apparatus with top coat of claim 1 to 2, it is characterized in that: described plastic basic unit is a kind of in culture dish, culture plate, the culturing bottle.
4. method that is used for cell cultures is characterized in that: cell seeding is cultivated on the arbitrary described cell culture apparatus with top coat of claim 1 ~ 3, made cell can keep a kind of more near form and function in the body.
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| WO2016070454A1 (en) * | 2014-11-06 | 2016-05-12 | 徐小杨 | Automation device, carrier and method for oocyte in vitro fertilization and cleavage culture |
| CN108148756A (en) * | 2016-12-05 | 2018-06-12 | 中国科学院大连化学物理研究所 | A kind of preparation method of low adherency culture plate |
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