CN1249778A

CN1249778A - Cell-culture system

Info

Publication number: CN1249778A
Application number: CN98803114A
Authority: CN
Inventors: P·L·布罗特; D·普勒
Original assignee: Smith and Nephew PLC
Current assignee: Smith and Nephew PLC
Priority date: 1997-01-16
Filing date: 1998-01-14
Publication date: 2000-04-05
Also published as: WO1998031783A1; CA2277815A1; AU5670698A; AU732565B2; GB9700840D0; JP2001509023A; ZA98329B; EP0954561A1

Abstract

A cell culture substrate device comprising a frame and a cell culture substrate material is disclosed. The frame is adapted to releasably secure the substrate material in preferably a flat configuration for the attachment of cells. The substrate material may further comprise a support layer. The device may be placed within a container, e.g. Petri-dish.

Description

Cell culture system

The present invention relates to be used for the substrate and the container of cell culture technology.Especially, the present invention relates to cell can grow thereon in order to next to transfer to the patient's who needs cellular layer substrate.

WO 91/13638 has described a kind of wound dressings, and it comprises a polymer layer, has the mammalian cell layer through cultivating that anchors to a surface.Such dressing is suitable for the processing of segment thickness wound, for example burns and supplies the skin position.These cells preferably can be from the epithelial cell of cultivating or cultivating from other source from the intrinsic chrotoplast of body that obtains by living tissue excision.They are through cultivating and growth in the presence of cell institute adhered polymer substrate.When this substrate is coated to the patient purchase the position time, cell is transferred on one's body the patient from substrate, and continuously breeding forms epithelium layer.Being prepared as follows of described dressing: by contacting culturing cell with polymerization dressing substrate surface; Remove substrate from substratum; Then dressing is applied to the patient so that cells contacting patient wound face.

One object of the present invention is to provide a kind of cell cultures substrate device of improved form, and this device conveniently is used for cell cultures and cell is anchored to substrate, and next cell transfer is arrived the patient.Another object of the present invention provides a kind of cell culture system that can realize above-mentioned purpose equally.

According to the present invention, cell cultures substrate device comprises the framework that is formed by pair cell nontoxicity or hypotoxic material and pair cell does not have toxicity and have at least a cell can adhere to the cell cultures substrate on surface thereon, described framework is fit to releasedly with the fixing described cell cultures substrate of suitable configuration, with cell adhesion on described at least one surface.

This framework preferably has suitable shape and big or small to be fit to cell culture container.Although other shape is suitable equally, preferred form generally is square, rectangle or circle.

This framework be fit to suitable configuration fixed cell culture substrate in order to cell adhesion to its surface.The cell cultures substrate preferably includes the polymeric film on the surface with suitable cell adhesion.This film can be hydrophilic, also can be hydrophobic.This substrate can comprise the material that one deck is above.

Suitable cell cultures substrate comprises those that mention among the WO 91/13638, be the adulterant or the multipolymer of polyester, polypropylene, vinyl acetate, for example ethylene-vinyl acetate copolymer, polyvinylidene dichloride, polystyrene, polyhutadiene, polyethylene, ionomer, SURLYN (trade mark) for example, or contain the multipolymer and the adulterant of these compounds.

For example the hydrophilic polymer of Hdyrophilic polyurethane also can form culture substrate.This culture substrate has mammalian cell through cultivating can adhere to thereon first surface.This surface can be treated to improve its performance, as another material coating with for example collagen base material, or by alternate manner, for example corona discharge or plasma treatment.This substrate is polymeric film preferably.Preferred this substrate and body profile can be fitted.This substrate can be a successive, also can be perforated.

The cell cultures substrate preferably is suitable for wound, with as wound dressings.Therefore the cell cultures substrate preferably can be fitted and be flexible, so that consistent with its coated wound profile.This material can be suitable for sucking.When the cell cultures substrate has when adhering to its surperficial epithelial cell, it can be coated on the wound, so that cell contacts with wound, form again to promote the wound epithelium effectively cell is transferred to wound from substrate.

When substrate comprised the plane material of polymeric film for example, this framework preferably was fit to flat basically configuration immobilized substrate.Cell converging on substrate can be reduced to minimum by this way, so that cell can be distributed on their adherent surfaces relatively equably.Therefore this framework preferably includes planar section, and this planar section is engaged to the described cell cultures substrate of small part, is being on the parallel plane with plane configuration immobilized substrate releasedly with described planar section.This planar section can comprise the successive layers of open-frame or material.In a preferred form, this planar section comprises open-frame, and it is that the cell cultures substrate of enclosed band can adhere to framework thereon that this open-frame has periphery.

This cell cultures substrate can by for example bonding, lamination, heating or radio-frequency welding or for example the mechanical system of folder to be fixed on the framework releasedly.Combination between framework and the cell cultures substrate is preferably without leakage.It can be that another surface of container or cell culture container internal surface goes to press part or all of substrate substrate is remained flat configuration relatively that this framework also can be used for.

This framework can also comprise the upstanding wall that can or cannot extend continuously around described planar section.This wall can comprise described framework is remained on parts in the container.For example, this wall can comprise the peristoma or the recess at the edge of the container that can engage culture dish for example or cell culture container.The other parts of preferred this wall and framework are an integral body.When wall is continuously and surrounds the planar section of framework, and the cell cultures substrate is when combining with framework, and framework and substrate can form the container that cell can be cultivated therein together.

Framework is by the pair cell nontoxicity or do not have the material of overt toxicity to make.Suitable material comprises polyester (for example polyethylene terephthalate, Polyethylene Naphthalate), comprises their adulterant and multipolymer, polyolefine, polystyrene, polycarbonate, acrylic substance, polyetherimide, ethylene-vinyl acetate copolymer and contains adulterant and multipolymer or other suitable material of these materials arbitrarily.This framework can be made by molded, for example blowing, thermoforming, vacuum or extrusion forming or injection molding.This framework can by polymeric film by vacuum forming and the heating to form desired shape.

Preferred this device is fit to be fixed in the container of culture dish for example.Therefore according to the present invention, cell culture system comprises a device, this device comprises framework and the pair cell nontoxicity that is formed by pair cell nontoxicity or hypotoxic material and has the cell cultures substrate that at least one cell can adhere to surface thereon, described framework is fit to fix described cell cultures substrate with suitable configuration releasedly, be used for cell adhesion to described surface and container, wherein said framework has definite shape and size being fit to described container, so that an internal surface of described cell cultures substrate and described container keeping parallelism basically.

This culture substrate can also comprise carrier layer.This culture substrate can be to stick on its part or all of lip-deep carrier layer releasedly.The surface that the preferred vector layer adheres to thereon is relative with a cell adherent surface.Under this form, by framework with plane configuration fixedly culture substrate and carrier layer.This carrier layer preferably includes the polymer film harder than culture substrate, and it is smooth that it helps keeping culture substrate like this, and is no wrinkling.This carrier layer can comprise and the suitable similar material film of material that forms framework.This carrier layer preferably stick to releasedly with respect to cell the whole surface of residue culture substrate on adherent surface.This carrier preferably bonds as follows: it can be removed by peeling off from the residue culture substrate when needs.

In a preferred form of the invention, the cell cultures substrate comprises that the mammalian cell through cultivating can the adherent first surface and second apparent surface, on most of at least second surface it with the first surface that adheres to the continuous polymer film that forms the culture substrate carrier layer releasedly on.

In one embodiment, the second surface of cell cultures is with on the part that adheres to carrier layer releasedly, the preferred center part.

The carrier layer remainder that does not contact with the opposing second surface of substrate can be to adhere on the framework releasedly or regularly.

According to a further aspect in the invention, a kind of cell cultures substrate device is provided, it includes framework that pair cell nontoxicity or hypotoxic material form and pair cell nontoxicity and has the cell cultures substrate that at least one cell can adhere to surface thereon, wherein a part of substrate is to adhere to releasedly on substrate another part, and described another part adheres on the described framework regularly.

Further describe the present invention referring now to accompanying drawing by embodiment, wherein:

Fig. 1 is the skeleton view according to apparatus of the present invention;

Fig. 2 is the cross-sectional view that is placed on the device shown in Figure 1 in the container;

Fig. 3 is the cross-sectional view of second embodiment;

Fig. 4 is the skeleton view that is placed on the 3rd embodiment device in the container;

Fig. 5 is the cross-sectional view according to Fig. 4 device.

Fig. 1 has shown a kind of cell culture apparatus, comprises framework 10 and cell cultures substrate 14.Framework 10 comprises the open type peripheral frame 11 that forms the frame plane part, and upstanding wall 13.Wall 13 and peripheral frame 11 integrally form by vacuum forming, and it is the polyethylene terephthalate film thin slice of thick 200m.Wall 13 has the peristoma 12 of extending around its upper limb.Cell cultures substrate film 14 by lamination with on the low surface that is fixed to peripheral frame 11 releasedly.The cell cultures substrate comprises the fitted thin slice of ethylene-vinyl acetate film, and the surface is handled to improve its surface property by corona discharge technology and adhered in order to reach optimum cell thereon.The centre portions of this upper surface can enter by the opening portion of peripheral frame 11.The framework and the culture substrate that form this device can be placed in the culture dish shown in Fig. 2 15.Preferred framework and culture substrate is aseptic and in case bacterial packaging supply.

In order to use this cell culture apparatus, it is placed in culture dish or other container, add then and contain for example Keratinocytic cell culture medium of human body epithelium.Many days of 37 ℃ of following culturing cells, cell proliferation and adhering on the substrate in the meantime was up to the degree of converging that reaches required cell density and cellular layer.Remove substratum then and shift out this device from container.Can from framework, peel off the cell cultures substrate then, and by placing it on the wound that will handle under the cell side direction.

In Fig. 3, culture substrate is hanged down surface adhesion on the carrier layer of the PET of thick 150m film thin slice.This carrier is given the hardness of increase to film 14, is beneficial to it is kept flat and does not have the corrugated situation.When cell growth and when adhering on the culture substrate, carrier and substrate are removed and are placed on one's body the patient from framework together, can remove carrier layer by peeling off thereon.

In Figure 4 and 5, on the part carrier layer 16 that limits by peripheral frame 11, cell cultures substrate 14 is strippingly to hang down surface adhesion on carrier layer 16 with it.To peel off in order being beneficial to, to provide label segment 17 towards substrate 14 corners to substrate 14.The remainder of carrier layer 16 adheres on the framework 11 regularly.

Claims

1. cell cultures substrate device, comprise the framework that forms by pair cell nontoxicity or hypotoxic material and pair cell nontoxicity and have the cell cultures substrate that at least one cell can adhere to surface thereon, wherein said framework is fit to releasedly with the fixing described culture substrate of suitable configuration, with cell adhesion to described at least one surface.

2. the device of claim 1, wherein culture substrate comprises the polymkeric substance on the surface with suitable adherent cell.

3. claim 1 or 2 device, wherein substrate comprises film.

4. according to the device of claim 2 or 3, wherein substrate is selected from polyester, polypropylene, vinyl acetate copolymer, polyvinylidene dichloride, polystyrene, polyhutadiene, polyethylene ionomer.

5. according to the device of above-mentioned arbitrary claim, its middle frame is fit to the fixing culture substrate of plane configuration basically.

6. according to the device of claim 5, its middle frame comprises the planar section that engages at least a portion cell cultures substrate, releasedly substrate is fixed with plane configuration being on the parallel plane with planar section.

7. according to the device of claim 5, its middle frame is the open-frame that comprises peripheral frame.

8. according to the device of claim 6 or 7, its middle frame also comprises the upstanding wall that extends around planar section.

9. device according to Claim 8, its mesospore also comprise framework are remained on parts in the container.

10. device according to Claim 8, the parts that wherein are used to keep comprise peristoma or the recess that engages container edge.

11. according to the device of above-mentioned arbitrary claim, wherein culture substrate by bonding to be fixed on the framework releasedly.

12. according to the device of above-mentioned arbitrary claim, wherein the cell cultures substrate also comprise with adhere to releasedly with at least one surperficial facing surfaces on carrier layer.

13. according to the device of claim 12, wherein the apparent surface covers a part of carrier layer.

14. according to the device of claim 13, wherein the apparent surface has covered the centre portions of carrier layer basically, limits the unlapped peripheral part in outside whereby.

15. according to the device of claim 14, wherein the unlapped peripheral part in outside adheres on the framework regularly.

16. the device of claim 1, wherein at least one surface also comprises label segment.

17. according to the device of above-mentioned arbitrary claim, and a collection of mammalian cell.

18. purposes according to the device of above-mentioned arbitrary claim.

19. cell culture system, it comprises a device, this device comprises the framework that formed by pair cell nontoxicity or hypotoxic material and pair cell nontoxicity and has the cell cultures substrate that at least one cell can adhere to surface thereon, described framework is fit to releasedly with the fixing described cell cultures substrate of suitable configuration, with with cell adhesion on described surface and container, wherein said framework has definite shape and size, to be fit to described container, so that described cell cultures substrate keeps parallel with an internal surface of described container basically.