EP0687185A1

EP0687185A1 - Process for producing an implant from cell cultures

Info

Publication number: EP0687185A1
Application number: EP94908950A
Authority: EP
Inventors: Michael Sittinger; Jesus Bujia
Original assignee: Individual
Current assignee: Individual
Priority date: 1993-03-03
Filing date: 1994-03-03
Publication date: 1995-12-20
Also published as: JP3567268B2; JPH08511679A; DE4306661A1; US5891455A; WO1994020151A1; DE4306661C2

Abstract

The process attempts to produce especially cargilage cells in vitro. The cells are placed in a three-dimensional supporting structure, preferably of a polymer nonwoven, with the shape of the supporting structure corresponding to that of the implant to be obtained. The supporting structure is then perfused with a nourishing solution for a long period so that the intercellular matrix bonding the cells together can form at least partially within the supporting structure. The supporting structure with the at least partially formed intercellular matrix is then implanted. On the subsequent resorption of the supporting structure, the shape of the implant is preserved thanks to the intercellular matrix then formed.

Description

i

Process for producing an implant from cell cultures

The invention relates to a method for producing an implant from cell cultures, in particular an implant with cartilage cells, these cells being applied to a resorbable carrier structure and then being implanted.

Should the body's own tissue be removed, e.g. Cartilage tissue is replaced, so far there are essentially two options:

1. The required tissue can be removed from corpses, preserved and then implanted. In some cases, immune reactions occur.

2. To replace the removed tissue, the patient's own tissue can be used, which is removed from the patient at a different location that is not visible from the outside and implanted at the location of the removed tissue. No immune reactions are to be feared here.

A decisive disadvantage of these two methods, however, is that, due to the ever increasing number of implantations, there is too little material overall, so that the desired replacement of the tissue is often incomplete.

A third method has therefore been proposed, namely cells which have been isolated and propagated in a conventional manner on a polymer fiber bundle made of resorbable material apply and implant the bundles; see. CAVacanti et al. , Plastic and Reconstructive Surgery, Vol. 8, No. 5, Nov. 1991, pp. 753-759. By stacking the bundles on top of and next to each other, the implant could be modeled three-dimensionally. After the implantation, the polymer material is resorbed, and at the same time the intercellular matrix, in particular collagen, is to be built up between the individual cells, so that in the final stage, a fully functional tissue structure is integrated into the surrounding tissue.

This method is still in the experimental stage, tested on animals, but has not yet been used in human medicine.

Difficulties with this method should arise when modeling the desired implant, since the shape of the fiber bundles placed one above the other and next to one another is not to be expected during the resorption of the fiber material. In addition, larger implants would have difficulty feeding the individual cells located inside the implant.

The invention is based on the object of specifying a method with which cell tissue, in particular cartilage tissue, is made available in a configuration which is favorable for implantation. In particular, the implant should have good formability and shape retention in the implanted state and ensure the nutrition of the individual cells in the implant.

This object is achieved according to the invention by the characterizing features specified in claim 1.

The basic idea of the invention is accordingly a three-dimensional, stable support structure with the desired implant shape made of a material with a coherent inner surface and a small one Volume, for example a polymer fleece, to introduce cells into the inner cavity of the carrier structure and to flow through the carrier structure receiving the cells with a nutrient solution. As soon as the intercellular matrix has at least partially formed, the entire three-dimensional support structure can be implanted.

The three-dimensional support structure ensures that the cells with their intercellular matrix remain dimensionally stable even during absorption and that the desired shape of the implant is maintained even after the implantation.

Perfusion ensures that the cells located within the support structure are also adequately supplied with nutrient solution and that this supply is maintained when the intercellular matrix has at least partially built up. As a result of the structure of the support structure and, if appropriate, a corresponding preparation, the nutrient solution enters the support structure and penetrates it essentially only by diffusion, which also corresponds to the situation in vivo. The nourishment of the cells accommodated in the carrier structure by diffusion can also be ensured by coating the entire carrier structure with a material that essentially only allows diffusion. Such a material is e.g. Agarose.

By feeding the cells in the carrier structure via diffusion, it is prevented that the cell products, in particular the collagens and proteoglycans necessary for the construction of the intercellular matrix, are washed out of the cell structure. The retention of these necessary factors, ie their retention, can be improved and supported if the support structures are perfused with the nutrient solution at intervals. The cell products are then on site during the perfusion breaks held and form the connections with the cells, whereas in the perfusion phases also undesired cell products can be washed out of the carrier structure and the matrix.

Further embodiments of the invention emerge from the subclaims.

The invention is explained in more detail in an exemplary embodiment with reference to the figure, in which the method is shown schematically with regard to the devices used.

The description only refers to the production of cartilage tissue in vitro; however, it goes without saying that other cell tissues can also be treated accordingly.

For implantation, the patient's own cartilage cells are grown in a conventional manner on a culture medium.

A carrier structure is produced from a resorbable, dimensionally stable polymer fiber fleece, the shape of which corresponds to the later implant. Suitable nonwoven materials are e.g. Polyglycols or polylactides. From this, nonwovens can be produced that have a large inner coherent surface with a minimal material volume. The continuous inner surface of the support structure is covered with an adhesion factor, e.g. Poly-L-lysine coated or wetted. This wetting takes place e.g. by immersing the support structure in a polylysine solution and subsequent lyophilization, i.e. Freeze drying so that the adhesion factor covers essentially the entire inner surface of the support structure.

If there is a sufficient number of cells in the above-mentioned cell culture, these will be correspondingly three-dimensional in the suspension with the culture medium prepared carrier structure filled. To increase the viscosity of the suspension, a resorbable, viscosity-increasing substance, for example the agarose mentioned, can be added.

The carrier structure receiving the cell is then coated with agarose, e.g. the support structure is immersed in a 2% agarose solution. The support structure is then subjected to a cold shock, e.g. by immersion in a cold water bath at about 4 ° C., whereby the agarose of the jacket and also the agarose, if any, added to the suspension solidify. Such a measure facilitates the following handling.

This carrier structure prepared in this way is designated 1 in the figure and is inserted into a perfusion apparatus 2. This apparatus 2 consists of a housing 3, in which a perfusion chamber 4 is provided, into which the carrier structure is inserted. A feed line 5 for a nutrient solution opens into the housing 3 and initially enters a mixing chamber 6, in which the flow is evened out before it enters the perfusion chamber 4. An outflow chamber 7 then adjoins the perfusion chamber 4, from which an outflow line 8 leads to a collecting container 9. The housing 3 of the perfusion chamber 4 is covered with a cover 10 and is kept at a constant temperature corresponding to the mean body temperature of 37 ° C. by a heater 11.

The actual perfusion chamber 4 can still be at least partially filled with agarose.

A nutrient solution, for example the solution commercially available under the name Hams F12, is slowly passed through the perfusion chamber 4. This nutrient solution is transported by a pump 12, for example a peristaltic pump, which is arranged in the feed line 5 and nutrient solution from one Sucks reservoir 13. This reservoir 13 is located, for example, in a water bath 14 at a temperature of 4 ° C. The amount of time that the peristaltic pump 12 is transported is very small and, in the present case, is set at approximately one milliliter per hour for the production of cartilage tissue. Some support structures - three such support structures 1 in the perfusion chamber 4 are shown in the drawing - can thus be sufficiently nourished, these support structures having a cross-section of at most one thumb nail size.

The peristaltic pump 12 is controlled at intervals by a timer 15, the intervals being adjustable depending on the size of the support structures and the perfusion chamber: in practice, the pumping and pausing intervals will be in the minute or hour range. Good results were achieved at pump and break intervals of approx. 30 minutes each.

Perfusion devices are known per se. They are otherwise used to grow cell cultures that are attached to a membrane with continuous conditions, thus replacing the conventional petri dishes. The membranes with the applied cell cultures can be inserted through slots into the perfusion chamber.

The nutrient solution passed through the perfusion apparatus 2 diffuses through the agarose jacket into the carrier structure 1, so that the cells taken up therein are fed. The flow of the nutrient solution and the interval control are set so that the cell products necessary for the construction of the intercellular matrix are not washed away, but remain in place. Over time, the intercellular matrix with its collagen fibers forms between the cells, so that the cells are bound together. The support structure and the agarose can at least partially dissolve during this long process. Due to the formation of the intercellular matrix, however, the predetermined shape and thus also the desired shape of the later implant is retained.

If the intercellular matrix is sufficiently developed, which is the case from about 10 days to a few weeks, the support structure is removed from the perfusion chamber and implanted. Over time, the matrix continues to develop as the support structure is reabsorbed.

It could be shown that such a method actually builds the intercellular matrix in vitro, which has not been observed to date, as far as is known.

It is also possible to add so-called tissue orphogenic factors to the fleece of the support structure. Such morphogenic factors stimulate cell formation and the formation of the intercellular matrix and thus cartilage formation. Such factors can either be introduced into the support structure during the formation of the intercellular matrix in the perfusion chamber or after the implantation into the support structure. An elegant solution is to couple the tissue-morphogenic factor directly to the nonwoven material or to an antibody and to provide the nonwoven material of the carrier structure with haptens to which the antibodies attach. As a result, these factors only act locally in the area of the support structure.

Claims

1. A method for producing an implant from cell cultures, in particular cartilage cells, the cells being applied to a resorbable support structure and then implanted together with the latter, characterized by the following features:

a three-dimensional, essentially dimensionally stable support structure preformed according to the desired shape of the implant is used with a coherent inner surface and a small volume;

the cells are introduced into the inner cavity of the support structure;

the carrier structure receiving the cells is perfused with a nutrient solution at least as long, i.e. flows through until an intercellular matrix binding the cells to one another has formed, after which the carrier structure can be implanted.

2. The method according to claim 1, characterized in that the carrier structure is a nonwoven made of polymer fibers, in particular polyglycols or polylactides.

3. The method according to claim 1 or 2, characterized in that the carrier structure, before the cells are introduced into it, is provided with adhesion factors, in particular poly-L-lysine.

4. The method according to claim 3, characterized in that the adhesion factors are applied by lyophilization to the iϊinere surface of the support structure.

5. The method according to any one of the preceding claims, characterized in that the inner cavity of the support structure is designed and dimensioned so that the nutrient solution diffuses through the support structure.

6. The method according to claim 5, characterized in that the carrier structure receiving the cells is coated with a material, in particular agarose, through which the nutrient solution can diffuse.

7. The method according to any one of the preceding claims, characterized in that the carrier structure with the cells accommodated therein is flowed through at intervals, so that there is a pause after each perfusion phase.

8. The method according to any one of the preceding claims, characterized in that the carrier structure with the cells accommodated therein is used for perfusion in a perfusion chamber.

9. The method according to any one of the preceding claims, characterized in that the cells in the suspension with the culture medium are introduced into the support structure, the suspension being additionally mixed with a viscosity-increasing substance, preferably agarose.

10. The method according to any one of the preceding claims, characterized in that a tissue morphogenic factor is added to the support structure, which supports the cell formation and the formation of an intercellular matrix.

11. The method according to claim 10, characterized in that the tissue-morphogenic factor is coupled to an antibody which attaches to the material of the support structure, preferably to haptens applied there.