CN1910272A - Composition and methods for cell culturing and tissue culture platforms - Google Patents

Abstract

This present invention describes a method of coating a polymer surface with diamond-like carbon (DLC) to render it useful as a carrier for cells derived from neural crest origin, preferable neuronal cells that form dendrites. The biopolymer to be coated with the DLC will include biodegradable polymers and other implantable biopolymers to act as a carrier system for cell transplantation into the various parts of the body, including the brain, the eye, the central and peripheral nervous system, the lung, the liver, the spleen, the kidney, and the bone and cartilage. The biopolymer can be in sheet form or microparticle form, and can be imbedded with, or incorporated into during its synthesis, attachment or growth promoting reagents to enhance and support neuraonal call attachment and growth. This coating method can also augment other coating agents such as extracellular matrix (ECM) secreted by cultured bovine corneal endothelial cells, as well as adhesive molecules such as fibronectin, laminin, and RGDS. The coating step can be a sequential process where the DLC layer will be aded on top of an ECM coated surface or an attachment factor coated surface.

Description

The composition and the method that are used for cell cultures and tissue culture platform

The application requires in the U.S. Patent Application Serial 60/510,358 of submission on October 10th, 2003 and 60/510,348 right of priority, and its full content is added the present invention as a reference.

Background of invention

Invention field

The present invention relates generally to by the improved method of using the various mammalian cells of cell culture method growth in vitro and the new cell culturing surfaces composition and the method for application.

The description of prior art

Advised that described treatment need comprise the disease treatment in eye, brain, liver, skin, cartilage and the blood vessel with the alternative approach of Transplanted cells as all organ replacements that are used for various treatment needs.For example see JP Vacanti etc., J.Pediat.Surg., Vol.23,1988, pp.3-9; PAebischer etc., Brain Res.Vol.488,1998, pp.364-368; CB Weinberg and E.Bell, Science, Vol.231,1986 pp.397-400; IV Yannas, CollagenIII, ME Nimni, ed., CRC Press, Boca Raton, 1988; GL Bumgardner etc., Hepatology, Vol.8,1988, pp.1158-1161; AM Sun etc., Appl.Bioch.Biotech., Vol.10,1984, pp.87-99; AA Demetriou etc., Proc.Nat.Acad.Sci.USA, Vol.83,1986, pp.7475-7479; WT Green Jr., Clin.Orth.Rel.Res., Vol 124.1977, pp.237-250; CA Vacanti etc., J.Plas.Reconstr.Surg., 1991; 88:753-9; PA Lucas etc., J.Biomed.Mat.Res., Vol.24,1990, pp.901-911.The ability that produces the human cell line in tissue culture can improve Transplanted cells as the prospect of treatment model with recovery forfeiture function of organization.It is very important can producing and produce people's cultured cells system from the neural crest tissue, usually can not self-regeneration from damage after individuality reaches the Adulthood because be derived from its tissue or organ.

Traditional be used for that tissue culture labware that cells in vitro cultivates is applied usually the propagation of negative charge when strengthening the adhesion of cultivating mammalian cell and some.But, with conventional tissue culture surfaces realize mammalian nervous unit cell gratifying adhesion, to keep and breed be the most difficult traditionally.By adding collagen gel layer or on tissue culturing plate and ware, promoting neural cell adhesion and propagation to improve the extrtacellular matrix deposition of rat EHS tumor cell secretion.Yet, hindered these The Application of Technology owing to existing material to have to before inoculating cell, be taped against at once this shortcoming of culture surface.

Use that the biological polymer carrier is supported to adhere to, growth, and finally in migration process, carry these cells as carrier, for cell replace treatment, the special success that the cell at the impaired usually brain that is derived from neural crest and eye rear portion is replaced treatment in aging course is important.The biological polymer that has 7 kinds of general categorys: polynucleotide class, polyamide-based, polyose, polyisoprene class, lignin, polyphosphoric acid salt/ester class and poly-hydroxyalkanoic acid salt/ester class.See for example United States Patent (USP) 6,495,152.The scope of biological polymer comprises from IV collagen to polysiloxane composition, its surface is embedded with carbon granule, perhaps it is handled by primary amine and optional peptide, perhaps it is by primary amine or contain the silane or the siloxanes co-processing (United States Patent (USP) 4 of carboxyl, 822,741), perhaps for example, the collagen of other modification is the known natural cartilage material (United States Patent (USP) 6 that contains, 676,969), described natural cartilage material has experienced degreasing and other processing and only remaining II Collagen Type VI material and glycosaminoglycan, perhaps alternatively the fiber of the II Collagen Type VI of purifying is mixed with glycosaminoglycan and any other desired additives.These extra additives are passable, for example, comprise and be used for assisting the chondrocyte the chondronectin of the adhesive attraction of II type collagen fiber or ankyrin II and somatomedin cartilage-inducing factor (CIF) for example, rhIGF-1 (IGF) and transforming growth factor (TGF β).

Before the present invention occurred, it was impossible cultivating Mammals or people's nervous tissue and make its growth in vitro and division from neural crest or individual neurone.

The invention summary

One aspect of the present invention discloses the method with the stabilizing layer coating tissue culture labware of carbon blood plasma, described carbon blood plasma most preferably is DLC, and its preprepared supply (ready supply) that can strengthen neural cell adhesion and growth and instrument can be provided is successfully to carry out the tissue culture of these cell types.

Knownly present two kinds of disadvantageous states from described neural crest source or particularly neuronic people or mammalian cell.The one, they do not duplicate usually in vivo or under conditions of tissue culture, and the 2nd, they are not very good to the adhesion of conventional cell culture surface.By with being called as the carbon blood plasma coating surface of diamond sample carbon (DLC), the inventor has been found that neurone can easily adhere to the culture surface and present propagation and replys.

These characteristics of the machinery of DLC film and tribological property and diamond very similar (airborne frictional coefficient is approximately 0.1, and hardness reaches about 80GPa, and Young's modulus is near 600GPa).In addition, these films are inert chemically in the most rodent environment, and can be to approach the density deposition of diamond density.But, to compare with the carbon gas deposition, diamond, DLC film at room temperature produce usually, and this makes them have a great attraction in the application that can't experience the pyritous base material.

The invention discloses the DLC coating to the deposition on biological polymer surface, it is backer and mammalian nervous unit and the adhesion and the growth that are derived from other cell type of neural crest successively again.

An object of the present invention is to produce the tissue culture platform of a specialization that is used for the growth in vitro of neuronal cell and neural crest source cell and keeps, to be used for the cell line proliferation purpose and to test.The product of the present invention of DLC coating comprises tissue culture ware, flask, slide glass, filter chamber, polymkeric substance and granulated glass sphere, plate, and piece.Described coating can be deposited on plastics, glass, synthetic and the natural biological polymer and metal.Described DLC coating can be added to the coating of other type for example by the improved product that is used for specific people or mammalian cell growth on the top of secreted extracellular matrix (ECM), adhesion molecule coating and somatomedin coating of the bovine corneal inner skin cell of cultivating with generation.

In addition, biological polymer used in the present invention can be natural or synthetic source.Natural biological copolymer comprises collagen and other known polymeric material.For the synthetic polymkeric substance, they can be acrylic acid and derivative or multipolymer polymethylmethacrylate for example, poly--N-N-isopropylacrylamide or poly--2-hydroxyl-metacrylate, polyvinyl alcohol and derivative and multipolymer.Described biological polymer can be thin plate or microparticle form.Support characteristic for the growth that strengthens described biological polymer, adhesion or positive growth factor can be embedded in building-up process or be incorporated in its composition.In addition, being suitable for supporting containing the growth of neurocyte of semi-solid biological polymer and the three dimensional growth medium that duplicates also can be coated with to improve it by DLC and support neure growth and the ability of keeping.Described biological polymer can also contain chitosan or Sodium Alginate these " maypolymer ".

By to following detailed description of the preferred embodiments, these and other target of the present invention, with and many benefits of following, it is brighter and clearer to become.

Detailed Description Of The Invention

When describing the preferred embodiments of the invention, for the sake of clarity adopted specific glossology.But the present invention also is not intended to be limit by these selected specific terms, and should be appreciated that each specific term comprises that all operate the technical equivalents that realizes similar purpose in a similar manner.

Method described in the invention allows with DLC and similar coating polymer surfaces to be coated with, thereby makes it can be used as the carrier derived from the cell in neural crest source.Biological polymer can be biodegradable part.Biological polymer can be with thin plate, microparticle, and perhaps semi-solid piece form exists.Use the blood plasma rifle to be coated with biological polymer, described blood plasma rifle will be the carbon blood plasma thin layer of 200-400  at purpose culture surface deposition one layer thickness.

The same with diamond sample (DLC) coating, decolorizing carbon nitride (C-N) film can be very hard and wear-resistant.They can be used as the problem that material standed for solves aseptic lax (aseptic loosening) in whole hip replacements.Du etc. have reported with scanning electron microscope study silicon and morphology amorphous C-N film sedimentary silicon on the performance of the scleroblast in the organ cultures in silicon, DLC coating.Cell on the silicon wafer (silicon wafers) can adhere to, but can not expansion after adhering to.On the contrary, on DLC coating and amorphous C-N film, then there is not obvious damage cell adhesion, expansion and propagation pair cell physiology aspect.The morphology of cell on coating and film is grown with the cell in contrast similar.Such result supports the DLC biological compatibility of coating, and is inspirer (C.Du etc., Biomaterials.1998 Apr-May to the potential source biomolecule medical use of amorphous C-N film among the present invention; 19 (7-9): 651-8).

The DLC coating process is as follows:

The blood plasma device is made up of following part: by Lawrence Berkeley NationalLaboratory, and the vacuum arc blood plasma rifle that Berkeley, CA produce, thus it operates in the repetition pulse mode high electric power and thermal load associated problem is minimized.The blood plasma rifle that is equipped with the carbon negative pole has formed the dense feathering (plume) of pure carbon blood plasma, and it has the oriented flow energy (streaming energy) of about 20eV.Blood plasma is injected into one 90 ° magneticstrainer (crooked solenoid) to remove all particulate materials from negative pole, subsequently it was transported one and be used for the big permanent magnetic porous configuration that graduation radiation shape blood plasma distributes.In this way, can make carbon blood plasma be deposited on the big deposition region scope internal space is homogeneous.

Homogeneity for further reinforcing membrane will be positioned on the dish of slow rotation by the matrix that DLC is coated with, thereby eliminate the position angle unhomogeneity.It is about 2-4000  that described device is used to form thickness, the DLC film of preferably about 200-400 .

In order to improve growth of biological polymer sustenticular cell or adherent ability, the adhesion mixture that contains one or more following materials can be embedded into or be incorporated in its composition in building-up process: concentration range is the fibronectin of 1 μ g-500 μ g/ml polymer latex, concentration range is the ln of 1 μ g-500 μ g/ml polymer latex, concentration range is the RGDS of 0.1 μ g-100 μ g/ml polymer latex, concentration range has been puting together of the 1ng-500ng/ml polymer latex bFGF of polycarbophil (polycarbophil), the concentration range EGF of polycarbophil that has been puting together of 10ng-1000ng/ml polymer latex, concentration range is that the NGF and the concentration of 1ng-1000ng/ml polymer latex is that scope is the heparin sulfate of 1 μ g-500 μ g/ml polymer latex.

In thin plate or microparticle form, in a preferred embodiment, the biological polymer of coating is used as carrier who is used for nerve growth and the carrier that is used for the cell transmission in the Transplanted cells step.Semi-solid polymer blocks form can be used as the function that the neurocyte holdout device is brought into play the nerve stimulation monitor with integrated circuit (IC) chip or the coupling of CCD chip.Can further improve the surface of coating by also spreading the DLC coating subsequently with the sedimentary extracellular matrix coating of the bovine corneal inner skin cell of cultivating.

Embodiment 1: with the biological polymer of DLC coated panel form

The biological polymer plate can be a virtually any size, and the biological polymer plate that preferably the present invention is of a size of about 2cm * 2cm is fixed on the rolling disc, and this dish is built up in the top of a slow rotation motor in the DLC application chamber successively.This blood plasma equipment will be by having about 20eV oriented flow can spray gun produce the dense feathering of pure carbon blood plasma.Blood plasma is injected in 90 ° of magneticstrainers to remove the diamond sample carbon that all particulate materials form high quality, no hydrogen.When by the big permanent magnetic porous configuration of transhipment by being used for that graduation radiation shape blood plasma distributes the time, carbon blood plasma deposition will spatially be homogenized in a very big deposition region.When carbon blood plasma feathering when holding the dish of slow rotation of polymer sheet, the uniform films of DLC will be coated with the surface of this plate.This plate can be used to grow cell of numerous species, and neurocyte preferably, itself or in UV radiation or 70% alcohol wash and the carrier of back of sterilizing as Transplanted cells.

Embodiment 2: with the biological polymer of DLC coating with the microparticle form

The biological polymer microparticle is placed in the rotating room of specialization the feathering of carbon blood plasma such generation as described in Example 1.When the rotating room slowly rotates with vertical axis, the blood plasma rifle is used to the DLC spraying is introduced in the chamber, like this, thus microparticle thrown the bottom continuously from the top and made carbon blood plasma have an opportunity the to be deposited to all surfaces zone of each microballoon in uniform mode.This process is guaranteed all even covering of finishing whole particle surfaces by lasting about 2-3 hour time.

Embodiment 3: will adhere in building-up process or positive growth factor embeds or is incorporated in the composition of biological polymer and is coated with DLC subsequently

In building-up process, adhesion or positive growth factor can be embedded or be incorporated in the composition of biological polymer of the present invention, the described factor comprises one or more in following: concentration range is the fibronectin of 1 μ g-500 μ g/ml polymer latex, concentration range is the ln of 1 μ g-500 μ g/ml polymer latex, concentration range is the RGDS of 0.1 μ g-100 μ g/ml polymer latex, the concentration range bFGF of polycarbophil that has been puting together of 1ng-500ng/ml polymer latex, the concentration range EGF of polycarbophil that has been puting together of 10ng-1000ng/ml polymer latex, concentration range is that the NGF and the concentration of 1ng-1000ng/ml polymer latex is that scope is the heparin sulfate of 1 μ g-500 μ g/ml polymer latex.Biological polymer is prepared into thin plate or semi-solid piece (bloc) subsequently, as described in front embodiment 1, realizes the deposition of DLC.Perhaps polymer manufacture can be become microparticle or spheroid, with the deposition that realizes DLC as described in the front embodiment 2.

Embodiment 4: also use DLC coated panel or microparticle subsequently with the sedimentary extracellular matrix coating of the bovine corneal inner skin cell of cultivating biological polymer

Before DLC is deposited to the culture surface, can at first use extracellular matrix (ECM) coating biological polymer plate and microparticle piece.In order to realize this purpose, (about 2000-150,000 cell/ml, preferably approximately 20,000 cells/ml) are inoculated into the surface of plate or piece, perhaps allow it to adhere to the surface of microparticle with low density with bovine corneal inner skin cell (BCE).The BCE cell is remained in the substratum that contains DME-H16, and described substratum is supplemented with 10% bovine serum, 5% foetal calf serum, 2% dextran (40,000MV) and the bFGF of 50ng/ml.Cell was cultivated 7 days among 10% the CO2 down at 37 ℃, during every other day add the bFGF that concentration is 50ng/ml.Remove the BCE cell by handling 5 minutes described polymer sheets, piece or microparticles with 20mM ammonium hydroxide.Subsequently, the PBS with capacity cleans the biological polymer that has the extracellular matrix coating for 10 times.After the drying, in embodiment 1, like that polymer sheet or the piece that is coated with ECM carried out the DLC deposition as described previously, and in embodiment 2, like that the microparticle that is coated with ECM is carried out the DLC deposition as described previously.After order was coated with ECM and DLC, described polymer sheet, piece or microparticle were sterilized by UV radiation or alcohol wash, and were used for nerve growth or as the carrier of Transplanted cells.

Embodiment 5: comprise the matrix of biological polymer, described biological polymer has the neurone that is electrically connected to unicircuit

In building-up process, adhesion or positive growth factor can be embedded or be incorporated in the composition of biological polymer of the present invention, the described factor comprises one or more in following: concentration range is the fibronectin of 1 μ g-500 μ g/ml polymer latex, concentration range is the ln of 1 μ g-500 μ g/ml polymer latex, concentration range is the RGDS of 0.1 μ g-100 μ g/ml polymer latex, the concentration range bFGF of polycarbophil that has been puting together of 1ng-500ng/ml polymer latex, the concentration range EGF of polycarbophil that has been puting together of 10ng-1000ng/ml polymer latex, concentration range is that the NGF and the concentration of 1ng-1000ng/ml polymer latex is that scope is the heparin sulfate of 1 μ g-500 μ g/ml polymer latex.Biological polymer is prepared into thin plate or semi-solid piece subsequently, as described in front embodiment 1, realizes the deposition of DLC.Perhaps polymer manufacture can be become microparticle or spheroid, as described in front embodiment 2, realize the deposition of DLC.

On the matrix of DLC coating, unicircuit or chip are in place.As be described in Zeck, G.﹠amp; Fromherz, Proc.Nat.Acad.Sci., 98,10457-10462 is described in (2001).Neurocyte will be placed on the silicon chip with DLC coating, and these neurocyte are enclosed by little continuously connected fastener (peg) original place subsequently.Near cell will grow into and be connected with each other and link to each other with this sheet.Stimulator under each neurocyte will generation can cause the voltage change of the electricimpulse that passes cell.The electricimpulse that puts on sheet will pass to another from a neurocyte, and passes sheet back to close (trip) transwitch.

Embodiment 6: in the DLC on the culture surface of tissue culture labware deposition

When flat culture surface of use such as ware, filter liner (filter insert), the chamber slide glass in the time of plate and piece, can up be placed on these utensil culture surface and present to the blood plasma rifle in the vacuum chamber, and be coated with operation as previously mentioned.When using microcarrier bead, they are flowed to guarantee that its all faces all are coated with equably in the chamber.For confining surface for example flask or pipe, a kind of blood plasma rifle of special modified can be inserted in this container and be coated with the purpose surface.Thickness is about 4000  of about 20-, and the uniform DLC thin layer of preferably about 200-400  will be deposited to the culture surface.Wash the product of sterilizing with UV radiation or alcohol subsequently, packing, sealing and storage are put on the shelf until use.

Embodiment 7: be coated with culture surface and DLC deposition subsequently in proper order with the bovine corneal inner skin cell excretory ECM that cultivates

In this embodiment, sparse culture (sparse culture) (about 1000-about 50 with bovine corneal inner skin cell, 000 cell/ml, preferred 2000-5000 cell/ml) is inoculated into the culture surface of required labware, and described utensil comprises ware, flask, pipe, filter liner, chamber slide glass, microcarrier bead, roller bottle (roller bottle), cell harvestor, sheet and piece.Cell is remained in the substratum that contains DME-H16, and described substratum is supplemented with 10% bovine serum, 5% foetal calf serum, 2% dextran (40,000MV) and the bFGF of 50ng/ml.Bovine corneal inner skin cell is cultivated 7-10 days until converging, during every other day add the bFGF that concentration is 50ng/ml.Remove substratum then, handled cell 3-30 minute with the distilled water solution of sufficient 20mM ammonium hydroxide.Subsequently,, dry in aseptic layer stream stink cupboard with the PBS clean surface of capacity 10 times to remove residual ammonium hydroxide.Carry out the coating of DLC subsequently as previously mentioned at the top of extracellular matrix.Wash the product of sterilizing with UV radiation or alcohol subsequently, packing, sealing and storage are put on the shelf until use.

Embodiment 8:DLC deposition back order adheres to or growth stimulant to the culture surface coated

In this alternate embodiment, one or more adhesions or growth stimulant comprise that concentration range is 1 μ g-500 μ g/ml fibronectin, concentration range is 1 μ g-500 μ g/ml ln, concentration range is 0.1 μ g-100 μ g/mlRGDS, concentration range is the bFGF that 1ng-400ng/ml has puted together polycarbophil, and concentration range is the EGF that 10ng-1000ng/ml has puted together polycarbophil.Described adhesion or growth stimulant will be added to the culture surface, and subsequently 4 ℃ of following incubations 20 minutes-2 hours.Subsequently with PBS clean surface 3 times and dry in aseptic layer stream stink cupboard.Subsequently in the top of lip-deep adhesion of culture or growth stimulant coating DLC layer deposited product.Then wash the labware of sterilizing with UV radiation or alcohol, packing, sealing and storage are put on the shelf until use.

Invention has been described, to those skilled in the art, is easy to the present invention is made many modifications and need not to leave the purport of the present invention that is limited by claims.

At this United States Patent (USP) that will above quote, the content of patent application and all other reference adds this specification sheets as a reference in full.

Claims (40)

1. one kind is used for cell growth and adherent improved surface, and it comprises the biological polymer of the diamond sample carbon surface that is coated with high quality, no hydrogen.

2. the improved surface of claim 1, biological polymer wherein is biodegradable

3. the improved surface of claim 1, biological polymer wherein is the form of plate.

4. the improved surface of claim 1, biological polymer wherein is the form of microparticle.

5. neuronic method of growth in culture, it is included in the inoculation and the neurone of growing on the biological polymer of the diamond sample carbon surface that is coated with high quality, no hydrogen.

6. the method for claim 5, biological polymer wherein is biodegradable.

7. the method for claim 5, biological polymer wherein is the form of plate.

8. the method for claim 5, biological polymer wherein is the form of microparticle.

9. the improved surface of claim 1, biological polymer is wherein embedded or has been integrated into adhesive agent in its building-up process, this adhesive agent comprises following one or more: ln, fibronectin, RGDS, puted together polycarbophil bFGF, puted together the EGF of polycarbophil and heparin sulfate.

10. the neuronic method of growth in culture is inoculated and the growth neurone on the biological polymer that its method that is included in use claim 9 prepares.

11. an instrument that is used for detecting the neurocyte signal, it comprises:

A) biological polymer unit, it has be embedded into or be integrated into adhesive agent in its building-up process, described adhesive agent comprises following one or more: ln, fibronectin, RGDS, puted together polycarbophil bFGF, puted together the EGF of polycarbophil, with heparin sulfate or nerve growth factor, it is enough to make neural or neurocyte is transplanted to low density and is bred in the described unit and send nervous process;

B) integrated circuit (IC) chip or electric charge coupling devices, it has a kind of mechanism for described nervous process or dendron manufacturing electrical connection;

C) be used for measuring feeler mechanism from neuronic electrical signal; With

D) be used for the mechanism of described adhesive die attachment to feeler mechanism.

12. the instrument of claim 11, biological polymer unit wherein is independently.

13. the instrument of claim 11, biological polymer unit wherein is semisolid.

14. the instrument of claim 11, integrated circuit (IC) chip wherein or electric charge link coupled device in its building-up process by in its surface coated comprise the adhesive agent of one or more nerve growth factors or diamond sample carbon, to strengthen electrically contacting between nervous process or dendron and the described chip.

15. one kind is suitable for supporting nerve growth and the three dimensional growth medium that duplicates, it comprises the semi-solid biological polymer that can support neure growth.

16. the growth medium of claim 15, it also comprises " May Polymer ".

17. the growth medium of claim 16, wherein said " May Polymer " has been embedded into or has been integrated into adhesive agent in its building-up process, described adhesive agent comprises following one or more: ln, fibronectin, RGDS, puted together polycarbophil bFGF, puted together the EGF of polycarbophil, with heparin sulfate or nerve growth factor, it is enough to make neural or neurocyte is transplanted to low density and is bred in the described unit and send nervous process.

18. the growth medium of claim 17, the concentration of wherein having puted together the bFGF of polycarbophil or heparin sulfate is about 50 μ g/mL, the concentration of having puted together the NGF of polycarbophil or heparin sulfate is about 50 μ g/mL, and the concentration of ln is that the concentration of about 500 μ g/mL and RGDS is about 500 μ g/mL.

19. one kind is suitable for supporting nerve growth and the three dimensional growth medium that duplicates, it comprises can support semi-solid biological polymer neure growth, that be coated with diamond sample carbon.

20. the growth medium of claim 19, it also comprises " May Polymer ".

21. the growth medium of claim 20, wherein said " May Polymer " has been embedded into or has been integrated into adhesive agent in its building-up process, described adhesive agent comprises following one or more: ln, fibronectin, RGDS, puted together polycarbophil bFGF, puted together the EGF of polycarbophil, with heparin sulfate or nerve growth factor, it is enough to make neural or neurocyte is transplanted to low density and is bred in the described unit and send nervous process.

22. the growth medium of claim 21, wherein said biological polymer is made into the shape of pearl, plate or microparticle.

23. one kind with the method for neuron transplantation in the acceptor host, it comprises the growth medium that the purpose neurone is inoculated into claim 19, makes described neure growth to enough density, and the described neurone in described growth medium is implanted described host.

24. one kind is suitable for supporting nerve growth and the three dimensional growth medium that duplicates, it comprises can support semi-solid biological polymer neure growth, that be coated with BCE-ECM.

25. a method of making the growth medium of claim 24 comprises:

A) a herd of cattle corneal endothelium (BCE) cell that will be suitable in the developing medium of its growth is inoculated in the described three dimensional growth medium with low density;

B) described BCE cell being grown to converges; With

C) handle the described sufficiently long time of three dimensional growth medium to remove described cell with described medium sucking-off and with ammonium hydroxide.

26. one kind is suitable for supporting nerve growth and the three dimensional growth medium that duplicates, it comprises can support semi-solid biological polymer neure growth, that be coated with BCE-ECM and diamond sample carbon.

27. the growth medium of claim 26, it also contains " May Polymer ".

28. the growth medium of claim 27, wherein said " May Polymer " has been embedded into or has been integrated into adhesive agent in its building-up process, described adhesive agent comprises following one or more: ln, fibronectin, RGDS, puted together polycarbophil bFGF, puted together the EGF of polycarbophil, with heparin sulfate or nerve growth factor, it is enough to make neural or neurocyte is transplanted to low density and is bred in the described unit and send nervous process.

28. the growth medium of claim 26, wherein said biological polymer is made into the shape of pearl, plate or microparticle.

29. have the Laboratory Instruments that is suitable for inducing cell growth and adherent coating, this instrument has internal surface and outside surface, wherein internal surface is the surface that contacts with cell medium with cell, and the described internal surface of described instrument is scribbled the film of diamond sample coating.

30. the instrument of claim 29, it is selected from by Tissue Culture Dish, plate, tissue culture flasks, plate, bottle, slide glass, filter chamber, slide chamber, roller bottle, the group that collector and pipe are formed.

31. have the Laboratory Instruments that is suitable for inducing cell growth and adherent coating, it is included in, and the film with diamond sample coating is coated with described instrument at least a other coating.

32. the instrument of claim 31, coating wherein is an extracellular matrix.

33. the instrument of claim 32, coating wherein is BCE-ECM.

34. a coating is applicable to the method for inducing cell growth and adherent Laboratory Instruments, its film that comprises diamond sample coating is applied to internal surface.

35. the method for claim 34, its also comprise at first with at least a other coating for example BCE-ECM be coated with the internal surface of described instrument, subsequently with the coating of diamond sample coating.

36. the instrument of making according to the method for claim 34.

37. the instrument of making according to the method for claim 35.

38. one kind is used for cell growth and adherent improved surface, it comprises the synthetic biological polymer of the diamond sample carbon surface that is coated with high quality, no hydrogen.

39. the improved surface of claim 38, wherein the synthetic polymkeric substance is a for example polymethylmethacrylate of acrylate copolymer and derivative thereof or multipolymer, poly--N-N-isopropylacrylamide or poly--2-hydroxyl-metacrylate, perhaps polyvinyl alcohol and derivative thereof and multipolymer.