DE102004024834A1 - Apparatus for conducting a liquid-air culture of epithelium - Google Patents

Abstract

The present invention relates to an apparatus and a kit for performing a liquid-air culture of epithelium. Furthermore, the invention relates to a method for producing a collagen-coated matrix which is part of the device, the use of said device for the liquid-air culture of epithelium and a method for the liquid-air culture of epithelium.

Description

The The present invention relates to a device and a kit for execution a liquid-air culture of epithelium. Furthermore, the invention relates a method for producing a collagen-coated matrix, which is part of the device, the use of said device to the liquid-air culture of epithelium and a method for liquid-air culture of epithelium.

The biotechnological production of functional tissue in vitro is commonly called "tissue engineering ". So produced fabric has many applications, eg. B. in the Cell biological research on the structure and function of weaving, as Test system for Medicines and for the Transplantation.

The biotechnological production of epithelial tissue is in yours Applicability progressed differently. The production of Skin replacement, the z. B. in large burn wounds an important application is being intensively researched. In the In vitro generation of corneal tissue is attractive, but still little ready for use. Also for the production of respiratory epithelium may exist not yet covered.

In consists of some anatomical regions of the respiratory tract Need for replacement of defective or no longer existing respiratory Mucosa or respiratory epithelium. So currently exist no satisfactory therapeutic options for care long-stretched tracheal stenoses. The fast obstruction of the prosthesis lumen by ingrowing connective tissue to form new stenoses, the lack of tissue integration and the risk of rejection limit one long-term use of the present used alloplastic materials. Prerequisite for one successful tracheal replacement is the realization of a lining with functional respiratory epithelium.

A further indication for the use of tissue engineering for the production of respiratory Epithelium is in the occlusion of Septal perforations. The treatment of septum perforations is problematic. The operation is currently the method of choice but with a relatively poor chance of success. prosthetic closures with silicone obturators hardly influence the perforation-specific symptoms. Farther also appears the replacement of cleared mucous membrane in paranasal sinuses therapeutically useful. Another indication for one possible clinical use of in vitro produced respiratory epithelium in the ENT field lies in the field of reconstructive surgery of the larynx.

The prosthetic restoration of the described defects could occur if epithelial cells succeed on artificial, biocompatible To locate matrices and differentiate into functional epithelium, those after implantation alone or possibly in combination with other materials required for molding and warranty mechanical properties, adapted by the organism become.

Especially would be useful the generation and use of autologous equivalents of the epithelium, so that problems with rejection the grafts are avoided when not using autologous tissue arise. Artificial autologous tissue can then serve to structures in certain anatomical regions with defective or no longer existing epithelium to replace. there It is essential that the new tissue be approximately the same specific Functions fulfilled like that originally existing tissues. So the tissue has to be finely differentiated and may not be in the for Proliferation and growth essential dedifferentiated phase are located.

A possibility The generation of epithelium is involved in the culture of adherent epithelial cells covering medium, ie in a submerged culture. For example was suggested to bone marrow cells, but also skin cells on to cultivate a sterile nylon mesh precoated with collagen or incubated with fibroblasts that produce collagen (DE 37 515 19 / WO 87/06120).

Also Cells from the respiratory mucosa were already on collagen-coated However, matrices cultivated, but not to develop final differentiated epithelial cells (Ostwald et al., Laryngotrino Otology 2003, 82, 693-699).

From The submerged culture differs in the liquid-air culture in that the cells here at the interface be cultivated between medium and air, so that from below one Supply of medium possible is and is in contact with air from above.

The in vitro generation of skin equivalents generally takes place in the liquid-air culture on a collagen gel in which fibroblasts are incorporated. For this purpose, the collagen gel is poured, for example, on the polycarbonate membrane of a tissue culture insert. The collagen gel intended for transplantation is not very stable because it is not a foreign body, stable matrix, which would give it extra stability and tear resistance. Therefore, damage to the generated tissue often occurs during transplantation. Attempts have been made to solve these problems by attaching the collagen gel to an additionally generated collagen network (Yang et al., Tissue engineered artificial skin composed of dermis and epidermis, Artificial Organs, 2000, 24, 7-17).

Also for the Culture of cornea was shown to compare the liquid-air culture of this tissue to the submerged culture a denser packing of the cells and a stronger cellular differentiation caused so that the morphology of the cornea is improved (Richard et al., Curr Eye Res 1991, 10, 739-749).

The production of largely differentiated respiratory epithelium has so far only succeeded in liquid-air culture in connection with the colonization of fibroblasts (Rhee et al., Ann Otol Rhinol Laryngol 2001, 110, 1011-1016, Yamaguchi, Arch Otolaryngol Head Neck Surg 1996, 122, 649-654). Here, a cell culture inserts commercially available (Millicell ^® -CM, Millipore, Bedford, Massachusetts), for example the PTFE membrane coated with collagen and seeded with epithelial cells, wherein the coculture is investigated with fibroblasts. This culture method is based on Elsdale and Bard (J Cell Biol 1972, 54, 626-637). Instead of Milli Cell ^® -Inserts also called Transwell inserts are often (available from Corning Costar, High Wycombe, UK) used (eg, Davidson et al., Am J Physiol Lung Cell Mol Physiol, 2000, 279, L766-L778) , These transwells are made of polyester or polycarbonate, but they are also available already coated with collagen.

The So far, attempts have been made to generate functional respiratory Epithelium had above all the goal, finally differentiated respiratory To generate epithelial cells to address analytical issues the function of differentiated cells (ciliary beat, mucin production etc.) to clarify. The amount of epithelium produced is not critical for this, so the commercially available Systems for liquid-air culture were sufficient.

Epithelium, that was generated by methods known in the art, is limited to transplantation, because on the one hand problems with the stability of pure collagen gels, and on the other the previously known Matrices used for the liquid-air culture of epithelial cells strong restrictions in terms of size and texture subject to the matrix.

task The present invention is therefore, alternative devices to carry out to provide a liquid-air culture of epithelium, the one increased flexibility allow in the design of the matrix, so that epithelium gene gene not just to analyze cell or tissue functions, but can also be used for transplantation.

• a) auf einer für Medium permeablen Halterung aufliegt und mit dieser nicht fest verbunden ist oder
• b) schwimmfähig ist.

This object is achieved according to the invention by the provision of a device which is suitable for carrying out a liquid-air culture of epithelium. It comprises a culture vessel and a collagen-coated culture medium permeable biocompatible matrix, the device being designed so that the matrix is oriented horizontally and does not rest on the bottom of the culture vessel and the surface of the matrix is below the upper edge of the culture vessel, where the matrix is either

• a) rests on a medium-permeable holder and is not firmly connected to this or
• b) is buoyant.

When Epithelium is in the context of this invention epithelial tissue of mammals, preferred by humans or frequently used experimental animals such as mouse, rat or pig, that has contact with air in its physiological environment. The is for example in the skin or cornea, but also in respiratory Epithelium of the case. Respiratory epithelium is particularly preferred. The term epithelium closes This includes both tissue and isolated epithelial cells.

The liquid-air culture is particularly suitable for the culture (cultivation) and differentiation of epithelial cells that are in contact with air even in their physiological environment. According to a particular embodiment, these are respiratory epithelial cells. For the supply of the cells with medium on the basal side, contact with medium is necessary, so that the base on which the epithelial cells grow adhä, in this case, ie the collagen-coated matrix, must be aligned horizontally. In order to ensure the accessibility of culture medium from the bottom, the matrix must not rest on the bottom of the culture vessel, on the other hand, it must be permeable to culture medium. So it is necessary to align the matrix over the bottom of the culture vessel. Likewise, the surface of the matrix (ie, its top surface) must be below the top of the culture vessel to permit supply of culture medium that is in the culture vessel when using the device. The filling level of the medium in the liquid-air culture should preferably be about +1 to -1 millimeters (mm), preferably +0.2 to -0.2 mm, based on the surface area of the matrix. The surface of the matrix (ie its top side) should therefore lie in the liquid-air culture in the interface between air and culture medium, as described above.

In a preferred embodiment the invention is the holder on which rests the matrix, on the bottom of the culture vessel or is attached to its bottom. Alternatively, it is also conceivable that the holder is supported on the edge of the culture vessel or mounted on the upper edge.

The holder is in a preferred embodiment (see 1 ) an upwardly open base vessel 1 with a flat edge, on the edge of which is the matrix 10 rests. The basic vessel 1 is comparable in a preferred embodiment with a Petri dish. In addition to a round shape of the base, however, any other shape, such as square or rectangular, can be selected. The flat edge ensures the horizontal alignment of the matrix, which is necessary for the liquid-air culture. If the matrix has sufficient rigidity, it may be sufficient for the matrix to rest on at least three contact points on the edge of the base vessel. Preferably, however, the matrix lies on the entire edge of the base vessel 1 on.

It is preferred that over the entire edge of the base vessel 1 a frame 5 resting on the matrix. Alternatively, a frame 5 even only at some points, z. B. 3 . 4 or more points of support, resting on the matrix. The frame 5 Holds the matrix on the base vessel 1 solid, the matrix preferably between base vessel 1 and frame 5 is brought in that she is taut. The weight of the frame 5 may be enough to put the matrix on the base vessel 1 keep tight. Preferably, however, at least one bracket connects 4 the frame 5 and the base vessel 1 on the outside of frame 5 and base vessel 1 so that the integrity of the matrix does not pass through the bracket 4 is disturbed.

The clip 4 has two arms, one of which is outside the frame 5 encompasses and one the base vessel 1 , for example, by an opening mounted therein 3 , Of particular advantage are bends at the ends of the arms to the outside, through which the pushing of the clip into the opening 3 of the base vessel and on the frame 5 is relieved. Preferably, the clip exists 4 made of elastic stainless steel, it can "according to 1 even be turned. The stapling of base vessel 1 and frame 5 Can be done by handling the clamp (s) with stronger tweezers.

The term frame presupposes that the frame 5 In contrast to a cover, does not cover the entire surface (ie at its center), otherwise the accessibility of air to the surface of the matrix necessary for the liquid-air culture would be disturbed. Preferably, there is a large central opening 8th in the middle of the frame 5 , Other geometric and in particular asymmetrical shapes with any position of the opening 8th are explicitly included.

There are several possibilities for designing the frame 5 holding the matrix on the base vessel 1 to fix reversibly.

In its simplest form is the frame 5 - Preferably in a round base vessel 1 - a flat ring, in the shape similar to a large washer.

A preferred option, however, is that the frame 5 which, as described above the entire edge of the base vessel 1 rests on the matrix, on its outer side a downwardly angled edge 9 includes, after putting the frame 5 on the base vessel 1 rests against the outside of the base vessel. This edge 9 hinders the frame 5 on slipping. The edge 9 does not have to be on the entire edge of the base vessel 1 abut, a design of the edge 9 It is also possible that only individual stops are located at suitable locations and prevent slipping.

Under the conditions of the liquid-air culture, medium should be in the culture vessel whose fill level corresponds to the height of the matrix in the device. Because when putting the frame 5 on base vessel 1 and matrix under these conditions easily bubbles under the frame 5 be caught, it is preferable that at least one opening 6 in the frame 5 allows aspiration of such bubbles with a syringe through the matrix, the culture vessel including device can be kept slightly inclined to air bubbles under the opening 6 to move.

Alternatively, the frame 5 be so pronounced that it fixes the matrix exclusively or mainly characterized in that it on the outside of the base vessel 1 resting on the matrix. In this case, a slipping of the frame 5 for example, either by one at the top of the frame 5 attached, inward edge can be prevented after putting the frame 5 on the edge of the base vessel 1 rests on the matrix or by lugs on the side of the base vessel 1 are attached and further slide down the frame 5 prevent.

Also, the frame can 5 when he is at the Outside of the base vessel 1 rest on the bottom of the culture vessel, provided that the matrix is sufficiently large that it is between the base vessel 1 and frame 5 can be held. Of course, the frame also by the shape of the base vessel 1 that z. B. may taper on its outer side upwards, be prevented from further sliding down, the shape of the frame 5 the shape of the base vessel 1 must be adapted so that the frame 5 at the base vessel 1 can be present.

To exchange between the medium in the base vessel 1 and culture vessel, it is preferred that the wall of the base vessel 1 at least one opening 3 having. This opening preferably does not break through the upper edge of the base vessel 1 , The openings 3 are still suitable to the staple (s) 4, the base vessel 1 and frame connects / connect, anchor. With sufficient stability of the base vessel 1 it is not necessary that the basic vessel 1 having a bottom.

A other device that allows one Matrix for the liquid-air culture is horizontal above the ground and below the ground To hold the upper edge of the culture vessel, is characterized in that a holder a support surface for the matrix that includes a network is. This bearing surface ensures the Supplying the epithelium to be cultivated with medium from the bottom the matrix. Preferably, the network of the support surface is coarse mesh, so has a Mesh size of at least 50-500 μm.

Also this holder is preferably on the bottom of the culture vessel or is attached to its bottom. Preferably connect a central or at least three at the edge of the support surface mounted at a suitable location Support the bearing surface and the bottom of the culture vessel. Preferably is over At the edge of the matrix on a frame that holds this tight on the support surface. Of the Frame can do this by its weight, but it can by brackets or hooks with the support surface or the supports of the bracket be connected. This attachment is reversible.

In another embodiment The device is the matrix for liquid-air culture in the interface between Air and culture medium introduced by the matrix is buoyant.

The Matrix can therefore be buoyant be because they are partially coated with a hydrophobic layer is. The arrangement and size of the parts or the part of the matrix coated with the hydrophobic layer have to be, is to float the matrix horizontally on the medium is from the shape, size and other Design of the matrix dependent. in principle but it is e.g. possible, a central part in the center of gravity of the matrix or several parts at the edge of the matrix, evenly around the Focus of the matrix are arranged with the hydrophobic layer to coat. Just like that Is it possible, to cover the entire edge of the matrix. More options can be easily determined by the person skilled in the art.

The Matrix can also be buoyant in that they are at least a buoyant one body is connected or rests on it, the matrix with the buoyant body like that must be connected or so must be superimposed on him, that their top when swimming in culture medium in the interface between air and culture medium.

For example The matrix can be reversible with staples or hooks within one Styrofoam, foam or other buoyant material frame be stretched, but also individual buoyant bodies are for attachment suitable because the matrix is on the surface of the medium on its own spreads.

If the matrix rests on a buoyant body, so must this for medium be permeable. This can be ensured by the fact that the buoyant body a Network is. Preferably, this network is coarse mesh, so has a mesh size of at least 50-500 μm. The net can become buoyant be that, at least partially, but preferably completely, with a hydrophobic layer coated is. The hydrophobic layer that covers the matrix or the net, is preferably a wax or fat.

A Such hydrophobic layer can be applied by passing the Matrix or the network in a solution of wax and / or fat immersed in a hydrophobic volatile solvent, so that the Wax and / or grease after the solvent evaporates the threads, not however, the spaces between of the network. The solvent is preferably gasoline or hexane. A concentration of wax and / or fat in the solvent from 0.1-20% is particularly suitable. Particularly suitable fats are e.g. Paraffins with a melting point of 50-70 ° C.

One Advantage of the device, whose matrix is buoyant, is that for the liquid-air culture no exact adjustment of the filling level of the culture medium necessary is.

The matrix used in the device may be made of biodegradable or non-biodegradable, durable material or a mixture exist. The matrix preferably consists of biodegradable material. This biodegradable material may be poly-L-lactide (PLLA), polyhydroxybutyric acid (PHB), polyglycolic acid, hyaluronic acid, Goretex, or a mixture thereof. Some of these materials have already been shown to be suitable for the cultivation of respiratory mucosal cells (Ostwald et al., Laryngotrino Otology 2003, 82, 693-699).

The biopersistence the synthetic polymers PHB and PLLA are larger than those of collagens, what kind of certain applications may be beneficial. PLLA has a degradation time of about two years, the degradation of PHB in the organism lasts even longer.

Also stable Materials can for the matrix be suitable, e.g. Polyethylene, polyamide, polyester, polyurethane, Polypropylene or a mixture thereof.

Prefers The material of the matrix is not immunogenic, so when transplanted neither the risk of rejection there still has to be an immunosuppression. critical is, however, that the material is biocompatible, e.g. Not cytotoxic. A test for biocompatibility of matrix materials is in Ostwald et al. (see above). For matrices other than the one said materials may have biocompatibility e.g. with the one described there Test be found.

In a preferred embodiment the matrix used in the device is a network or network, wherein the mesh size of the network is preferably about 50-500 microns. Basically, however also a smaller mesh size of the network, up to about 0.2 or 0.4 μm possible, However, this is because of an unfavorable ratio of permeable surface to impermeable surface the network less expedient. The Meshes can be regular or irregular shaped be.

alternative could the permeability the matrix for The culture medium also be ensured by the fact that the Matrix porous is. For example, could a porous one Matrix from PLLA can be used (Lo et al., J Biomed Mater Res 1996, 30, 475-484). This material is particularly interesting in that it succeeds as a carrier with slow release of drugs (Zilberman et al., J Biomater Sci Polym Ed, 2002, 13, 1221-1240). Through targeted Drug delivery in vivo after transplantation could this Therefore, material is used to control specific processes, e.g. to advancement vascularization, suitable.

The devices described make it possible to use a matrix for the liquid-air culture, in which the size and shape of the surface are basically arbitrary. It can - in addition to a round or rectangular matrix - so for example, a form of the matrix can be selected, which already corresponds to the shape of a required graft. The area of the matrix is preferably greater than about 1 cm ² . For example, an area of about 4 cm ² is generally sufficient for the supply of Septumperforationen. However, the area can also be, for example, greater than about 500 mm ² or larger than a little 710 mm ² . Also, matrices whose area is larger than about 10,000 mm ² are usable with the described devices for the liquid-air culture.

The Device can - with a new matrix - reusable be. Therefore, the holder used in the device is made preferably from a sterilizable (e.g., autoclavable material) such as stainless steel, teflon or glass. Glass, like plastic, has the Advantage that a microscopic examination is possible in the culture of cells, if the culture vessel from a consists of these transparent materials.

The Culture vessel can in principle be every vessel that for the cell culture is suitable and adapted in shape and size to the holder, so it's bigger than the holder. A cell culture coating of the wall of the culture vessel is not necessary because the culture of the cells takes place on the matrix.

• a) die Matrix mit einer Kollagenlösung in Kontakt bringt,
• b) die Kollagenlösung antrocknen läßt,
• c) das Kollagen vernetzt,
• d) eine Behandlung mit einer Aminosäure- oder Proteinlösung durchführt und
• e) man die Matrix mit destilliertem Wasser wäscht.

The present invention also provides a process for producing the collagen-coated matrix for performing a liquid-air culture which is part of the described device. This method is characterized in that

• a) bringing the matrix into contact with a collagen solution,
• b) allowing the collagen solution to dry,
• c) crosslinking the collagen,
• d) performs a treatment with an amino acid or protein solution, and
• e) washing the matrix with distilled water.

The collagen solution preferably has a concentration of 1-10 mg / ml. For example can collagen G solution or collagen A solution (e.g., Biochrom-Berlin), but collagen G is prefers.

The crosslinking in step c is preferably a chemical crosslinking. This crosslinking can be carried out with mono- or dialdehyde, for example with glutaraldehyde. Alternatively, however, it is also possible to crosslink with physical methods, for example by irradiation or dehydration. Furthermore, an enzymatic crosslinking of the collagen or a copolymerization with Acrylderi be made ( DE 693 15 483 ). Crosslinking of collagen increases mechanical strength, therefore, the collagen-coated matrices used in this invention are preferably cross-linked.

The Crosslinking of collagen can lead to reactive groups arise. Before the matrices should be used for cell culture, should therefore, in step d, treatment with an amino acid or protein solution to saturation or neutralization of these reactive groups. The washing step e is used to remove unbound amino acids or Proteins and can with distilled water, but also with culture medium or other non-toxic solution, e.g. PBS, to be performed. Preferably, several washing steps are carried out.

Especially advantageous is a method in which the coating of the matrix carried out with collagen on a hydrophobic pad. This simplifies the subsequent supersede of the coated matrix as compared to peeling off the bottom of a cell culture dish or a similar one Vessel considerably and allows it thus, reproducible without damaging the collagen coating the matrix also larger coated Manufacture and use matrices.

The receives hydrophobic substrate It is preferred by a film of Wax, oil or paraffin. For this purpose, paraffin is preferably used with a melting temperature from 50-70 ° C. After the paraffin is liquefied by heat, you pour one thin layer (sterile) in a vessel suitable Size, e.g. Plastic Petri dishes for the cell culture, and let it cool. After a single use to coat a network with Collagen, the hydrophobic base is preferably discarded, on the one hand for reasons sterility, on the other hand because of possible Contamination in the following coatings due to residues of previous coatings. The storage on the hydrophobic base while The collagenization does not have harmful or cytotoxic effects the subsequent one Cell culture.

object The invention further relates to the use of the device described to the liquid-air culture of epithelium. As already described, sets the liquid-air culture precedes that culture medium to a level introduced into the culture vessel is where the surface is the matrix in the interface between culture medium and air.

Farther is a method for liquid-air culture of epithelium subject of present invention, in which uses a device described above becomes.

The Matrix of the device is used in this procedure with epithelial cells populated. Epithelial cells can can be obtained by known methods (Ross, et al., Eur Arch Otorhinolaryngol 1997, 254 (Suppl. 1), 12-17; Risbud et al., J. Biomed Mater Res 2001, 56, 120-127; Ziegelaar et al., Biomaterials 2002, 23, 1425-1438), for example by "outgrowth" or dissociation of cells of material, e.g. from septal corrections or conchotomies may or may have been taken for this purpose (e.g., "skinsnips") fabric pieces fixed on the matrix intended for fouling, proliferating cells populate the neighboring matrix. A dissociation of cells can e.g. with protease XIV or trypsin / EDTA solution, alternatively with 0.1% collagenase or 0.1% dispase or combinations of the enzymes.

In an embodiment The matrix is pre-populated with epithelial cells with fibroblasts populated. For the purposes of a later Transplantation is beneficial to fibroblasts of the transplant recipient use, i. autologous fibroblasts to prevent rejection reactions. Preferably both fibroblasts and epithelial cells are from the transplant recipient. in principle but it is also possible not to use autologous fibroblasts.

fibroblasts can be made out of one piece of fabric, which is also used for the production of epithelial cells enzymatic dissociation with 0.1% collagenase and subsequent cultivation in serum-containing medium (e.g., DMEM) in standard cell culture vessels cultivated (e.g., by the method of Gray et al., Am J Med Genet. 1987, 8, 521-526).

in the Unlike previous ones Method of liquid-air culture of epithelium (e.g., Yamaguchi et al., Supra) is it for a differentiation of the epithelium used in the process, with collagen-coated matrix not necessary that the matrix first with Fibroblasts is colonized. The colonization with fibroblasts is therefore optional.

Prefers The culture of epithelium is carried out after settling the matrix first a submerged culture and then a liquid-air culture. The submerged culture intensified the initial one Proliferation of epithelial cells. After the matrix with a sufficient Number of epithelial cells is colonized, depending on culture conditions and number of sown Cells after about 2-4 weeks, leads to have a liquid-air culture through, in which the differentiation of the epithelium takes place.

One particular advantage of the devices described is that they For this Change from submerged culture to liquid-air culture are suitable. The easiest possibility is, the matrix without first place the holder on the bottom of a culture vessel and after the submerged Culture to insert the matrix into the device. This possibility exists because the matrix is not irreversibly fixed in the device.

at Use of the described mounts on which the matrix is placed A submerged culture can also take place if so much medium is poured into the culture vessel, that the surface of the medium over the surface the matrix lies. This method has the advantage of being manipulated the matrix causing damage the collagen and / or cell layer can lead, largely avoided can be.

alternative could also - at constant height of Medium - the a Mount for the matrix may be formed so that the height of the matrix is adjustable, for example with supports, in two height settings can be locked.

at Use of buoyant Matrix there are several ways to reach a submerged culture. For one, the buoyant matrix with a weight, preferably at several points on the edge of the Matrix, be complained so that the matrix does not become the surface of the Culture medium can ascend. On the other hand - if the matrix thereby floatable is that she is connected with at least one buoyant body or on it is superimposed - this Compound or bedding produced only after the submerged culture become.

The Height of Culture medium in the culture vessel can at a discontinuous media change after visual inspection be set. However, a method is advantageous in which one for the Liquid-air culture is an automatic control device for the Height of Culture medium used in the culture vessel, which is designed so that the height of the culture medium on the height of Matrix is set. The use of such an apparatus allows a continuous exchange of medium and thus one optimal supply of nutrients.

In a preferred embodiment The invention is the epithelium respiratory epithelium, skin or Cornea. It is particularly preferred to include respiratory epithelium cultivating, for example epithelium from the nasal mucosa, septum, Larynx, gingiva, bronchi, or trachea.

Preferably the process of liquid-air culture of epithelium is carried out until the Epithelium differentiates so that functional epithelium is present, that for a transplant is suitable. In respiratory epithelium is this the case, for example, if at least 75% the area a layer of rounded, cobblestone-like epithelial cells has formed that too big Demonstrate part of microvilli, but prefer to recognize moving cilia to let.

object The invention also provides a kit comprising the described device, Instructions for liquid-air culture of epithelium and an appropriate amount of culture medium contains, optional in addition to Reuse the holder multiple replacement matrices.

Basically all culture media usual in the laboratory, e.g. RPMI 1640 (Roswell Park Memorial Institute), DMEM (Dulbecco's Modified Eagle Medium), IMDM (Iscove's Modified Dulbecco's Medium) also suitable for use in the liquid-air culture, DMEM is however preferred. It can be serum-free medium or with serum spiked medium (e.g., with 10% fetal calf serum (FCS)). However, the use of serum may lead to unwanted proliferation of fibroblasts lead, so serum-free medium is preferred. The medium may be, e.g. by the previous culture of fibroblasts, or be conditioned contain defined growth factors, e.g. EGF or insulin. The Use of antibiotic containing medium (e.g., penicillin / streptomycin or gentamycin) is preferred. Especially for the generation of differentiated respiratory epithelium is the combination DMEM / HamsF12 (about 3/1), with different additives (EGF, cholera toxin, insulin, hydrocortisone, transferrin, selenium, Bovine pituary extract, antibiotics (e.g., penicillin / streptomycin), preferred (Usui S et al., Ann Otol Rhinol Laryngol 2000, 109, 271-277).

• a) einem Patienten entnommenes Epithel auf der Matrix einer beschriebenen Vorrichtung ansiedelt,
• b) während einer Proliferationsphase eine submerse Kultur des Epithels durchführt,
• c) während einer Differenzierungsphase eine Liquid-Air-Kultur mit der beschriebenen Vorrichtung durchführt.

The invention further relates to a method for producing functional autologous artificial epithelium, wherein

• a) locating epithelium taken from a patient on the matrix of a described device,
• b) performing a submerged culture of the epithelium during a proliferative phase,
• c) performs a liquid-air culture with the described device during a differentiation phase.

Of the Patient can be both a human patient and a Be an animal.

The apparatus that can be used in the context of this invention for the automatic control of the height of the culture medium in the culture vessel, may include an electronic control of the level with medium (see, for example DE 198 01 763 ). However, it is advantageous to use an apparatus for regulating the height of the surface of medium in a culture vessel in which at least one inflow of medium is fed into a culture vessel and by at least one drain whose opening is located at the desired height of the surface of the medium, Medium is discharged (see. US 5,565,353 ).

In a preferred embodiment However, the apparatus is designed so that the desired height of the medium adjustable in the culture vessel is because of such special advantages, especially with regard to the desired Sequence of submerged and liquid-air culture of epithelial cells is reached can be.

object The invention also relates to a kit comprising the device for liquid-air culture of epithelial cells, the device being an automatic control device the height the surface of the medium, and in addition Instructions for implementation liquid-air culture and, optionally, multiple replacement matrices.

Compared to the Prior art, the present invention has many advantages on. The devices described, the matrix in the boundary layer between medium and air and thus a liquid-air culture allow, make it possible existing restrictions in terms of size and texture to overcome the matrix. Thus, epithelium can now be cultured in vitro, especially for the Transplant is suitable. The reversible connection of the matrix with the rest of the device it allows the overgrown matrix directly for one To use transplant.

It In the context of the present invention, it has been shown that it is suitable for execution The liquid-air culture is particularly advantageous, if the level of the medium in the culture vessel.

The The invention therefore further relates to a device for regulating the filling level of the medium in a culture vessel (control apparatus), at the desired filling level the medium is adjustable, as well as the use of this device for cell or tissue culture. Furthermore, the invention relates to a method to carry out a liquid-air culture using an inventive, hereinafter in more detail described device.

These Device is especially for perfusion culture (continuous Inflow and Outflow from Medium) and became more common on the basis Cell culture vessels developed. An automatic, continuous exchange of medium has over one discontinuous medium change with visual control at the Cell culture has several advantages, especially in longer culture times more often Make media change noticeable. An advantage is the optimal, constant supply of nutrients and the constant removal of toxic metabolic products dar. A continuous medium exchange allows a special dense culture of cells. A discontinuous medium change affected many especially sensitive cells are negative, since fluctuations in Nutrient concentration, pH, temperature and other factors may occur. The Cells become more subject to conditions with continuous exchange of medium cultured, which resemble physiological conditions.

One further advantage, especially in longer-lasting cultivations a continuous culture with defined media height in that by eliminating the frequent opening of the Culture vessel for discontinuous media change reduces the risk of contamination.

One third advantage of continuous culture as opposed to discontinuous Variant for longer Culture times is that the Workload is reduced.

One particular advantage arises in the cell culture, when replacing from medium to height the surface of the medium can be kept constant. This is discontinuous Medium change e.g. because of possible Evaporation is not always guaranteed. In principle, a constant height the surface the medium for Every cell culture important to maintain an optimal ratio of medium volume over the gas space allow the medium and thus an optimal gas exchange. For some special applications, e.g. the liquid-air culture, is one exact regulation of altitude but especially important.

An apparatus for automatic control of the level of the culture medium in the culture vessel may comprise an electronic control of the level with medium (cf., for example DE 198 01 763 ). However, it is simpler to use an apparatus for regulating the height of the surface of medium in a culture vessel in which medium is fed into a culture vessel through at least one inlet and through at least one outlet whose opening is at the desired level of the surface of the medium. Medium is discharged (see. US 5,565,353 ).

Problems arise, however, in the use of such an apparatus when the desired height of the surface of the medium should be variable, for example, to different culture conditions in a submerged and a subsequent Li Quid-air culture of epithelial cells, in which the cells are to be cultivated at the interface between medium and air.

With the provided and used in the context of the present invention Device in which the desired level of the medium in the culture vessel adjustable is, advantageously, the inventive liquid-air culture be performed.

Around the desired Height of level a culture vessel with Culture medium, a device is provided, which is a culture vessel for cell and tissue culture, the at least one inflow and at least one outlet for medium comprises being the height the drain opening (s) adjustable within the culture vessel is.

Under "culture vessel for cell and tissue culture " in the context of this application each vessel understood that to the culture of cells or tissues, preferably eukaryotic cells or tissues is. The culture vessel consists preferably of a material suitable for the culture of cells, in particular from a plastic coated for cell culture or Glass. Preferably, the culture vessel has the basic shape of a Petri dish, but also other forms, e.g. a rectangular plan or the shape of cell culture bottles is possible. The culture vessel should so executed be that a sterile culture of the cells is possible. For that should it is sterile lockable be. Preferred is the visual and microscopic control of Cell culture in the closed culture vessel possible. For this, the culture vessel must at least partly made of a transparent material, such as glass or plastic, consist. Into the culture vessel becomes, as described, the matrix for introduced the Liquid-Air culture.

Prefers comprises the culture vessel one Inflow and a drain with a drain opening. Are e.g. several outlets included, so must a control of the level at least the height the bottom drainage adjusted become. The height of several outflows of course also be adjusted together.

Under "Medium" is under this Registration every liquid understood in the vessel to culture is contained by cells, and whose fill height is to be adjusted. It is preferably a classic cell culture medium, such as. DMEM, RPMI medium, Iscove's medium, etc., if appropriate with additives such as. Fetal calf serum, Growth factors or antibiotics. Under medium, however, will another, in general for the cell culture compatible Liquid like e.g. PBS (Phosphate buffered saline) understood. Also solutions about to detach of cells, e.g. Trypsin / EDTA are within the scope of this invention referred to as medium, since the control apparatus also special for changing liquids suitable in the culture vessel is. Also, a gradual exchange of different culture media can be made with it.

Especially a device is used where the drain port is part a drain neck is. The drain hole is the opening the outlet nozzle, which is located inside the culture vessel. The spout is for passing liquids suitable. In general, the spout is a pipe or hose. Preferably, the outlet nozzle has a round inner cross section. The discharge opening is preferably at one end of the outlet, However, it can also be attached laterally with a closed end be. This may be useful if it is desired that the level of the medium is not arbitrarily adjustable, but e.g. always a certain minimum levels are available should. In this case, a part of the discharge nozzle as a spacer serve.

Of the outlet connection is preferably guided by a seal in a wall of the culture vessel. When Wall of the culture vessel becomes the entire shell of the Culture vessel denotes, So the side wall or side walls and the top and bottom of the culture vessel, i. also cover (as far as e.g. the top is designed as a removable lid) and bottom.

A Seal is located on the outlet and take care of a conclusion to the Wall of the culture vessel. A Seal therefore provides, as far as they are in use of the device for cell culture in the area that is not filled with medium, for a sterile conclusion to the outside of the culture vessel, the does not have to be sterile. If the seal is in the area filled with media, the seal must continue to prevent leakage of the medium, So one opposite form the medium tight seal. Preferably, the seal used is suitable for both purposes.

A simple form of such a seal is elastic and therefore lies both at the outlet connection as well as the culture vessel. Seals off Rubber or silicone are well known to those skilled in the art. Also a possible greased ground glass or similar However, it may be suitable, as far as a close to medium tight conclusion is required. The seal has a suitable size, so that the outlet pipe is movable in it.

In a preferred embodiment, the discharge nozzle is movably guided in the vertical direction through the lower or upper side of the culture vessel. Preferably, the discharge nozzle is through a Seal at the top of the culture vessel ge leads. The top of the culture vessel may be, for example, a Petri dish, a lid.

Prefers the spout protrudes from the culture vessel and over the Seal out. Then the outlet pipe and with it the height of the drain opening can be especially be easily moved without it would be necessary to open the vessel, the always associated with the risk of contamination. The length, um which protrudes the spout, is dependent on a vertical mobility of the discharge spout thereof, such as the height set the discharge opening is. The length, around the spout may protrude maximally or minimally from the culture vessel, such as far the height the level of the medium in the culture vessel adjusted can be should. This height is preferred freely adjustable. So drainpipe should be so long that it sticks out of the culture vessel and over the seal at every setting.

It may be useful, the spout, for example with a stop below and / or above the vessel wall or To design the seal so that an accidental too strong Slide in and / or complete Pull out the drain neck is prevented.

In an embodiment the seal is an elastic piece of tubing, with its lower end with a mounted on the top of the culture vessel holder connected is. This holder can be designed differently be possible e.g. an upwardly directed edge around an opening, onto which the tube piece is pushed can be. The holder can be made of the same material as the Culture vessel or else made of another suitable material. The holder can homogeneously drawn from the material of the top of the culture vessel its (e.g., glass) or the same or another suitable one Material purpose appropriately shaped and a hole in the top airtight (e.g., with silicone glue, epoxy glue). At the upper end of the hose piece this is located on the part the outlet nozzle, which protrudes from the culture vessel, at.

An apparatus comprising such a culture vessel is schematically shown in FIG 4 displayed.

To the same principle, but with an oblique support and guidance of the Drain spout, it is also possible the outlet through a side wall of the culture vessel, and preferably so, that the outlet nozzle from oblique protrudes into the top of the culture vessel.

alternative the device is designed so that the drain opening part of a discharge spout which is a non-moving one firmly attached to the culture vessel Part includes, which protrudes into the interior of the culture vessel, and a piece of tubing that as a movable part of the outlet nozzle is attached to the non-moving part and includes the drain opening. In this embodiment the apparatus, as in other possible embodiments, the seal does not have to be formed separately, as its function from the spout yourself is perceived. The height the discharge opening is adjustable, by the moving part more or less on the immovable Part is plugged. The height is in this embodiment not from the outside, but adjustable from within the culture vessel. This can For example, a sterile pair of tweezers can be used to control the sterility of the inside to ensure the vessel. If the vessel has a Removable lid, this can be removed for setting be, but also e.g. through the opening of a cell culture bottle is such an adjustment possible. Generally, plugging into a connected part is an Aufstek ken equivalent to a connected part, wherein a flow of the medium guaranteed have to be.

Preferably, the discharge nozzle protrudes from above or from below into the interior of the culture vessel. In this case, movable and immovable part of the downcomer should have a straight basic shape. Alternatively, the stationary part of the downcomer may protrude from a side wall into the interior of the culture vessel and be angled or bent, the movable part having a straight shape. An apparatus comprising such a culture vessel is schematically shown in FIG 5 displayed.

In an alternative embodiment is the drain opening part a drain spout, which is angled or bent and passed through a side wall of the culture vessel, being the height the drain opening a rotation of the outlet nozzle is adjustable. An angle of about 90 ° is preferred. If the outlet connection is guided by a seal and over the wall of the culture vessel and the seal protrudes, the height of the drain opening of Outside be set.

alternative can the drain opening part a drain neck be a straight, firmly attached to the culture vessel, non-moving Part includes, which protrudes into the interior of the culture vessel, as well an angled or bent piece of tubing, as a moving part of the drain neck the non-movable part is plugged and the drain opening comprises. Then is the height the drainage opening, as described above, e.g. with tweezers, by turning the movable part adjustable.

One wants to avoid that when using the control apparatus, the medium supplied interferes with the surface of the medium, which may be disadvantageous for a liquid-air culture, for example, or that medium injected during supply to side walls or top of the culture vessel, the culture vessel should be designed so that the inflow or the inflow opening is below the opening of the outflow, which determines the level of the culture vessel with medium. The inflow opening may be directly an opening in the wall of the culture vessel, optionally with a holder for a hose to be fastened or a pipe connection. However, the inflow opening may also be part of a Zuflußstutzens, which is guided through a wall of the culture vessel, optionally by a seal or firmly connected. Preferably, a Zuflußstutzen near the bottom of the vessel is guided by a seal in a side wall. A feeder, if, for example, a Petri dish or other standard culture vessel is used, can also be passed through the lid. While the spout is also routed through the lid, such a lid, which includes the effluent and influent essential to the invention, may be used in combination with any suitable bottom Petri dish or matching standard bottom.

object The invention also relates to a device, which further comprises a storage vessel with fresh Medium comprises, that connected to the inflow is, and further a collecting vessel includes old medium that connected to the drain is. When using the device, these vessels are preferred cooled. Alternatively, the storage vessel with fresh Medium but also e.g. at the desired temperature of the cell culture be kept if the cells are sensitive to temperature changes are. A collection vessel is not absolutely necessary, because the old medium, in contact with the Cells was, can also be discarded immediately.

The respective connection between inflow and outflow and storage vessel may e.g. a pipe joint, but are preferred because of their flexibility Hoses. The device comprises Thus, further preferred at least one hose connected to an inflow is, and at least one hose connected to a drain is. In particular, at least one device used on the device Silicone hose, preferably all hoses used.

object The invention also relates to a device which further comprises at least one Pump includes. One Pump can be the inflow of the Control medium, and a second pump, or the same pump, the outflow of the medium. Alternatively you can Inflow and outflow of the medium also e.g. be driven by gravity by the reservoir with new medium above the Culture vessel and the Collection vessel under arranged the culture vessel becomes.

In order to when using the device according to the invention the level of the medium in the culture vessel corresponds to the height of the outlet, may - after To fill of the vessel to desired Height - the amount supplied Media discharged the crowd Do not exceed medium otherwise the level does not remain constant, but rises. Therefore, the amount must inflowing and effluent Medium coordinated. After the first filling of the Culture vessel corresponds then the amount of the discharged Media automatically the amount of medium supplied, because of the Construction principle of the device no longer drained medium as supplied can be. The skilled person will easily, e.g. about the performance of the pumps and / or the inner diameter of the hoses, inflow opening (s), outflow opening (s), outlet connection etc., the amount of supplied or discharged Can set media so that not too much medium is supplied becomes.

Prefers are the inflow opening (s) and the drainage opening (s), the spout (s), Hoses and Pump power (s) of the device designed and successive agreed that the amount of the dischargeable medium at least as big as or is larger as the amount of feedable Medi ums. Preferably, the capacity of all outflows is greater than the capacity all tributaries.

Prefers are inflow and Outflow from Medium over coupled the same peristaltic pump. The operating principle of the peristaltic pump is based on the impression or squeezing a flexible pump hose to one or several places and by movement of the depressed spot in desired conveying direction the liquid. The movement of the oppressed Place is realized by means of a pump rotor, on its circumference itself cylindrical Rotor rollers are located. The rotor speed (movement speed the oppressed Position) and the inner diameter of the hose determine the delivery rate. For the same inner diameter of tubing, the same peristaltic pump spanned, is the production capacity that's the same.

Preferably, therefore, the inner diameter of the hose connected to the drain is at least as large as the inner diameter of the hose connected to the inflow. More preferably, the inner diameter of the tubing connected to the drain is greater than the inner diameter of the tubing connected to the tubing, since an inner diameter indicated as equal is often a problem since the diameters of the tubing are generally used as inflows or outflows , often not exactly are the same.

If the regulation of inflow or outflow via a peristaltic pump (or several peristaltic pumps with the same pump power), so Here, the diameter of the pump tubing in the hose pump is crucial, so here's the "pumping" hose a bit larger diameter as the "zupumpende" hose should have other hoses (e.g., from storage vessels to Pump, from the pump to culture vessel) should be possible a slightly larger diameter as the pump tubing to have.

The device according to the invention is suitable for use in cell and tissue culture. The invention therefore relates to the use of the apparatus for cell and tissue culture, as well as a method for cell or tissue culture, in which one carries out the culture in a device according to the invention. For culture of the cells, the culture vessel is preferably introduced into a suitable incubator whose temperature and CO ₂ content can be optimally adjusted for the cultured cells. The cell and tissue culture preferably comprises a liquid-air culture, especially the liquid-air culture of epithelial cells. In liquid-air culture, for example, the liquid-air culture of epithelium, the cells are attached to a matrix, which can be introduced into the boundary layer between medium and air. The cell bound or adhered to a matrix may also be cultured submerged, either on the bottom of the culture vessel or held by a device above the bottom of the culture vessel.

For the optimal Culture of tissue, e.g. to transplant, is the supply especially with nutrients important, so that the device according to the invention for this particular suitable is. Various pieces of tissue may be placed in a culture vessel, e.g. in a Petri dish, cultivated in different compartments is divided. These do not prevent the exchange of medium, enable however, the simultaneous cultivation of various defined fabric pieces without their mixing. Compartmented means that the bottom of the Through Stages or similar is split so that different samples in the resulting Compartments ("troughs") are placed can and do not mix during transport / movement of the culture vessel. The height The webs are held so that they are well below the height of the medium lies. The webs can be slidably mounted. The compartmentalization allows the comparative cultivation e.g. of cells that are based on matrices adhere to different materials or from different tissue samples under otherwise identical Conditions and allows the safe assignment of samples. Various Samples can also with the aid of several devices for introducing matrices for the Liquid-air culture to be cultivated in a culture vessel whose Fill level with medium over the controlled control apparatus is controlled.

The Devices illustrated in the drawings illustrate possible embodiments which Devices according to the invention are. however, not limited to these particular embodiments. Further Embodiments disclosed within the scope of the description are obvious to those skilled in the art seen.

List of pictures:

1 : Schematic of an apparatus for performing a liquid-air culture of epithelium with an open-topped basal vessel

2A : Apparatus for performing a liquid-air culture of epithelium with an open-topped basal vessel

The figure shows components of the device of 1 in a particular embodiment. On the left is the base vessel, to the right of which is the border to be placed on the matrix and the brackets on the front. At the rear, the device is shown in a composite form with a clamped matrix. The matrix was placed on the base vessel and the edge was securely fastened to it by means of the clips. In this form, the device can be introduced into the culture vessel with medium.

2 B : Device for carrying out a liquid-air culture of epithelium with a culture vessel, wherein the holder stands on the bottom of the culture vessel

The Illustration shows an open one Culture vessel, here a Petri dish into which a device for storage or storage of matrix materials with cells can be introduced. The above Pictured in the image device consists of a mesh stainless steel, which stands on metal feet.

3A : Proliferating fibroblasts on a collagenized matrix (material: polyamide, mesh size: 150μm)

3B : Proliferating respiratory epithelial cells on a collagenized matrix of the device according to the invention (material: polyamide, mesh opening: 150 μm)

In the following figures, which show the device for controlling the level of the medium in a culture vessel, the ver the following terms have the following meaning:
11 : Culture vessel, 12 : Inflow, 13 : Drain opening, 14 : movable part of a drain neck, 15 : non-moving part of a drain neck, 16 : Level of the medium in the culture vessel, 17 : Storage vessel with fresh medium, 18 : Collecting vessel for old medium, 19 : Lid of the culture vessel, 20 : elastic hose, 21 : Bracket, 22 / 22 ' : Drainage nozzle, 23 : Peristaltic pump

4 : Device for automatically controlling the level of the medium in a culture vessel, wherein the outlet nozzle is guided through a seal at the top of the culture vessel.

5 : Device for the automatic control of the height of the surface of the medium, wherein the outlet nozzle is guided by a seal on the side wall of the culture vessel, wherein a movable piece of tubing is pushed onto a non-movable, angled part of the discharge nozzle.

• A: Gezeigt sind die Hauptkomponenten einer Ausführungsform des Systems, Schlauchpumpe, Vorrats- und Ablaufgefäß (üblicherweise im Kühlschrank) sowie das Kulturgefäß mit Absaugung des Mediums von oben.
• B: Die Abbildung zeigt im Detail ein geöffnetes Kulturgefäß (Petrischale) mit Absaugung von oben. In dem Gefäß können entweder direkt oder nach Einbringung einer geeigneten Vorrichtung z.B. indirekt auf einer Matrix Zellen kultiviert werden. Die gezeigten Kammern in der Petrischale verhindern nicht den Austausch von Medium, erlauben jedoch eine Kultur definierter Zellen oder Gewebe in den verschiedenen Kammern.
• C: Gezeigt ist ein geöffnetes Kulturgefäß (Petrischale) mit Absaugung von unten, in die z.B. eine Vorrichtung zur Aufnahme bzw. Ablage von Matrixmaterialien mit Zellen eingebracht werden kann. Die oben im Bild abgebildete Vorrichtung besteht aus einem Siebgewebe aus nichtrostendem Stahl, welches auf Metallfüßen steht.

6 : Device for regulating the height of the surface of medium in a culture vessel

• A: Shown are the main components of one embodiment of the system, peristaltic pump, storage and drainage vessel (usually in the refrigerator) and the culture vessel with suction of the medium from above.
• B: The picture shows in detail an opened culture vessel (Petri dish) with extraction from above. In the vessel, cells can be cultured either directly or after introduction of a suitable device, for example indirectly on a matrix. The chambers shown in the Petri dish do not prevent the exchange of medium, but allow a culture of defined cells or tissues in the various chambers.
• C: Shown is an opened culture vessel (Petri dish) with suction from below into which eg a device for receiving or depositing matrix materials with cells can be introduced. The device pictured above in the picture consists of a stainless steel screen cloth resting on metal feet.

7 : Culture of fibroblasts under discontinuous (A) or continuous (B) media changes

1. coating a Matrix with collagen

Around to prepare the coating of the matrix became a base, e.g. a Petri dish, coated with a thin film of paraffin (melting point 50-70 ° C). It was for each attempt a new Petri dish (diameter 80-100 mm) thin with heated Paraffin poured out, which solidifies under the laminar box.

One sterilized network of a suitable polymer, e.g. made of polyamide (Reichelt Chemical Engineering or Institute of Polymer Research Dresden) was placed on the coated Petri dish and a solution of Collagen G (2 mg / ml in PBS (Phosphate buffered saline), Biochrom) given to it. The matrix was incubated at 37 ° C for at least 15 minutes, then the collagen solution sucked off and the collagen dried. The next step was the dried Layer covered with sterile 0.25 glutaraldehyde in PBS, immediately again sucked off and dried again. Finally, a satiate the still reactive aldehyde groups by covering the collagen layer with an amino acid solution (approx. 0.1 M, e.g. MEM-NEAS, Gibco / Invitrogen 100x, 1: 100 in PBS), followed immediately was sucked off again. Before use, it became so collagenized Rinse matrix at least 3 times with sterile distilled water.

Result:

The Matrix was without damage detached from the hydrophobic base and for the culture of epithelial cells used.

2. Coat a net with a hydrophobic layer

For the production a buoyant surface, placed on the one matrix for the liquid-air culture of epithelial cells a net of polyamide was transformed into a solution of Paraffin immersed in hexane, with different concentrations tested were. Before use, the evaporation of the solvent (at least about 30 minutes at RT) waited.

Result:

The Pad was able to withstand all concentrations tested to swim on culture medium. At concentrations of 0.2-20% wax in solvent were the strings The network of wax enclosed, however, the spaces between free. A matrix stored on the network had contact with medium and air, and cultured respiratory epithelial cells proliferated.

3rd extraction of epithelial cells

Human nasal mucosa explants from septal or nasal polyps were stored immediately after surgery for at least 30 minutes in gentamycin solution (100 μg / ml in PBS, Gibco) at 4 ° C. The tissue was divided into pieces of 3 mm x 3 mm. Subsequently, pri epithelial cells were obtained by enzymatic dissociation from the tissue or by fixation on the matrix and subsequent "outgrowth" of the cells.The cells were first cultured submerged, ie covered with culture medium (SFM or DMEM 10% FCS) (37 ° C., 5%). CO ₂ ).

Dissociation of the cell material 3.1 with protease XIV (Pronase ^®), or protease XIV (Pronase ^®) Dispase

The pieces of tissue were in Pronase ^® (0.1% in PBS, Sigma) or Pronase ^® and Dispase (each 0.1% in PBS) for about 16 hours at about 5 ° C and then about 90 minutes at 37 ° C shaken. The epithelial portion of the pieces of tissue was further processed with a scalpel to dislodge loosened epithelial cells from the cell structure. Subsequently, the cell suspension was washed twice with PBS and applied to the matrix to be grown.

3.2 Sequential dissociation of the cell material with trypsin / EDTA (Ethylenediamine Tetraacetic Acid)

The fabric pieces a total of four times in a solution of trypsin (0.5 μg / ml, Gibco) and EDTA (0.2 μg / ml) in PBS without calcium and magnesium ions (Gibco) at 37 ° C for 45 minutes shaken. The supernatant with the solved Cells were each centrifuged and incubated in DMEM (Gibco) with 10% FCS (Gibco). added. The recoverable fractions were collected, washed and applied to the matrix to be grown.

3.3 "outgrowing" the cells

Tissue pieces were applied to the matrix to be grown. They were fixed there by laying of sterile coverslips or by sticking of the tissue with fibrin glue (TissueColl ^® dui, Immuno, 1:10 in PBS).

Result:

The dissociation with pronase ^® or pronase and dispase ^® yielded compared to the sequential trypsinization a lesser amount of nichthämatogenen cells. However, was passed from the obtained by means of pronase ^® or pronase and dispase ^® cells each about 10% of the cells from respiratory epithelial cells with beating cilia, such that the dissociation of these enzymes in the sequence are preferably used. Since the yield of cells with beating cilia in the dissociation with these enzymes is approximately equal to, Dispase is not inactivated by fetal calf serum, the treatment was favored only with pronase ^®. Of the seeded cells, about 15-25% grew.

For the subsequent liquid-air culture, the highest possible density, but at least 100,000 dissociated cells / cm ² matrix is recommended. If this cell count is not achieved on the basis of limited sample material, intermediate cultivation in collagen-coated cell culture vessels (eg BD-Biocoat) should be carried out. The final mobilization of the cells should not be carried out with trypsin / EDTA but with gentler procedures (eg with Accutase, PAA Laboratories).

Also the "outgrowth" of cells from undissociated Fabric pieces, e.g. with laying on coverslips on the tissue, is possible.

Around a sufficient density of cells for the subsequent liquid-air culture Achieve is a distribution of tissue pieces at a distance from each other about 20 mm recommended.

4. Culture of epithelium

4.1 Submerse culture of Epithelium on slides

Since a submerged culture was to be investigated here, films of PLLA (poly-L-lactide) or PHB (polyhydroxybutyric acid) were colonized with or without prior amino functionalization of the films or collagenization with respiratory epithelial cells obtained by the methods described under 3. above , The culture took place under standard cell culture conditions (37 ° C, 5% CO ₂ ) with SFM or DMEM 10% FCS. The medium was changed intermittently for visual inspection.

Result:

Without Aminofunctionalization was poor, especially for PHB Observe attachment of cells. After about 3 weeks grew - on settlement suboptimal amount of tissue or cell suspension cultures of 2-3 cm diameter on the matrix. For several weeks culture developed complex three-dimensional structures with fibroblasts and epithelial cells on the matrix. Up to approx. 20 days after the start of the experiment, but afterwards no more, were loose and solid cells in the submerged culture with active cilia movement by light microscopy. It will therefore, these cells suspect tissue-derived cilia-bearing cells are therefore not proliferating or differentiating under culture conditions are.

Cells, when harvested from dissociated tissue, after 15-30 days, when harvested by "outgrowth" after 20-70 days, began to partially detach from the matrix and continue to grow as suspension cells at this time point the culture was ended.

4.2 Submerged and Liquid-Air Culture of epithelium on networks

Collagenized matrices made of networks of biocompatible polymers, eg of polyamide, were colonized with respiratory epithelial cells obtained by the methods described under 3.. The collagenized matrices had partially been previously colonized with fibroblasts. The culture took place under standard cell culture conditions (37 ° C, 5% CO ₂ ) with SFM or DMEM 10% FCS. The medium was changed intermittently for visual inspection. First, a submerged culture was carried out for about 14-21 days. Then the matrix was placed between the base vessel 1 and the frame 5 clamped a device already described and the height of the medium adjusted so that the surface of the matrix was only minimally covered with medium.

Result;

To Another 14-21 days of liquid-air culture, the matrix was dense covered with epithelial cells that also demonstrated microvilli. Cultures based on fibroblast-populated matrices showed better results. The main difference between submerged culture and liquid-air culture on matrices Networks lay in the higher Differentiation state of the epithelial cells. The liquid-air cultures but were also much denser.

5. Transplant of in vitro generated epithelium

From the nasal septum of rabbits tissue pieces can be removed under anesthesia. Using the methods described above, both fibroblasts and epithelial cells are recovered therefrom and placed sequentially on a reticulated collagen-coated polyamide matrix. For 14 days a submerse and then for a further three weeks a liquid-air culture is performed, in which the matrix between the base vessel 1 and the frame 5 clamped a device already described and the height of the medium is adjusted so that the surface of the matrix is only minimally covered with medium until the epithelial cells Microvilli and movable cilia show.

This Autologous graft can easily leave the device for liquid-air culture solved and used to put one again under anesthesia Defect in the nasal septum of the rabbit cover. The graft can be fixed by fibrin glue and / or suture.

6. Implementation of Culture using the level control device:

In Test series was found to be discontinuous at submerged cultivation of fibroblasts on collagenized mesh a media height of about 10 mm essential parameters of the medium (DMEM, 10% fetal Calf serum) already completely changed after 24 hours. So has after this time the content of glucose as the main energy supplier of about 5 mM reduced to about 0.2 mM, while the content of lactate from about 1.5 mM (from fetal calf serum) to about 9 mM. Usually In the case of such cultivations, the medium is not until after approx.

3 Changed days. In culture of fibroblasts with the device according to the invention lactate and glucose levels remained under continuous media changes but essentially stable.

In 7A and 7B are shown after the same culture fibroblasts, which were cultured on collagenized network (mesh size 150 microns). It can be seen that the cells from the culture with continuous exchange of the medium ( 7B ) are more dense - ie they have proliferated more strongly than in the case of discontinuous medium changes ( 7A ).

Claims (32)

Apparatus for conducting a liquid-air culture of epithelium comprising a culture vessel and a collagen-coated culture medium permeable biocompatible matrix 10 comprising, wherein the device is formed so that the matrix 10 is oriented horizontally and does not rest on the bottom of the culture vessel and the surface of the matrix 10 is below the upper edge of the culture vessel, characterized in that the matrix 10 either a) rests on a medium-permeable holder and is not firmly connected to this or b) is floatable. Device according to claim 1, characterized in that that the holder stands on the bottom of the culture vessel or on its bottom is appropriate. Device according to claims 1 and 2, characterized in that the holder is an upwardly open base vessel 1 with a flat edge, on its entire edge is the matrix 10 rests. Apparatus according to claim 3, characterized in that over the entire edge of the base vessel 1 a frame 5 on the matrix 10 rests. Device according to claim 4, characterized in that the matrix 10 tight between base vessel 1 and frame 5 lies. Device according to claims 4 and 5, characterized in that at least one clip 4 the frame 5 and the base vessel 1 on its outside connects. Device according to claims 3 to 6, characterized in that the wall of the base vessel 1 at least one opening 3 having. Device according to claims 1 and 2, characterized in that the holder has a bearing surface for the matrix 10 which is a network. Apparatus according to claim 8, characterized in that above the edge of the matrix 10 a frame rests. Device according to claim 1, characterized in that the matrix 10 is buoyant, the matrix 10 partially coated with a hydrophobic layer. Device according to claim 1, characterized in that the matrix 10 is buoyant, the matrix 10 is connected to at least one buoyant body or rests on a buoyant medium permeable body, wherein the matrix 10 connected to the at least one buoyant body or so deposited on it that the surface of the matrix 10 when swimming in culture medium in the interface between air and culture medium is aligned. Apparatus according to claim 11, characterized in that the buoyant body on which the matrix 10 rests, is a network that is coated with a hydrophobic layer. Apparatus according to claim 10 or 12, characterized that the hydrophobic layer is a wax or fat. Device according to claims 1 to 13, characterized in that the matrix 10 Made of biodegradable or non-biodegradable material or a mixture thereof. Device according to claim 14, characterized in that that the biodegradable material poly-L-lactide (PLLA), polyhydroxybutyric acid (PHB), polyglycolic acid, hyaluronic acid, Goretex or a mixture thereof and not biodegradable Material polyethylene, polyamide, polyester, polyurethane, polypropylene or a mixture thereof. Device according to claims 1 to 15, characterized in that the matrix 10 a network is. Device according to claim 19, characterized in that that the mesh size of the network is about 50-500 microns. Device according to claims 1 to 20, characterized in that the surface of the matrix 10 greater than about 100 mm ² , preferably greater than about 500 mm ² , in particular greater than about 710 mm ² . Process for the preparation of a collagen coated matrix 10 for carrying out a liquid-air culture of epithelium, characterized in that a) the matrix 10 b) the collagen solution is allowed to dry, c) the collagen crosslinks, d) a treatment with an amino acid or protein solution, and e) the matrix 10 washes with distilled water. Method according to claim 19, characterized the crosslinking in step c is a chemical crosslinking. Method according to claim 20, characterized in that that one carries out the crosslinking with glutaraldehyde. Process according to claims 19 to 21, characterized in that the coating of the matrix 10 with collagen on a hydrophobic pad. Method according to claim 22, characterized in that that one obtains the hydrophobic base, that one on a pad a movie of wax, oil or paraffin. Use of the device according to claims 1 to 18 for the liquid-air culture of epithelium. Process for liquid-air culture of epithelial cells, characterized in that the matrix 10 a device according to claims 1 to 18 populated with epithelial cells. A method according to claim 25, characterized in that the matrix 10 colonized with autologous fibroblasts before colonization with epithelial cells. Process according to claims 25 and 26, characterized in that after the colonization of the matrix 10 first a submerged culture and then do a liquid-air culture. A method according to claims 25 to 27, characterized in that one uses for the liquid-air culture, an apparatus for automatically controlling the height of the culture medium in the culture vessel, which is designed so that the height of the culture medium of the height of the matrix 10 equivalent. Process according to claims 25 to 28, characterized that the epithelium is respiratory epithelium, skin or cornea. Process according to claims 25 to 29, characterized that one carries out the culture, until the epithelium differentiates. Kit, characterized in that it is the device according to the claims 1 to 18 and a suitable amount of culture medium as well as instructions to carry out a liquid-air culture of epithelium. Process for the preparation of functional autologous artificial epithelium, characterized in that a) a patient-removed epithelium on the matrix 10 a device according to claims 1 to 18, b) performs a submerged culture of the epithelium during a proliferation phase, c) during a differentiation phase performs a liquid-air culture with a device according to claims 1 to 18.