DE10104008A1 - Growing and treating cells on a modelable structure, useful for making bioartificial tissue e.g. cartilage, where the cell culture space is subjected to alternating pressure - Google Patents

Abstract

Growing/treating cells on a modelable carrier structure (A) in which the cells, for producing a cell layer, are introduced into a cell culture space (7) (which is subjected to alternating pressure loading through a gas medium) between (A) (or a film applied over (A)) and a second, especially microporous, film, with oxygen and/or nutrients introduced into (7), is new. An independent claim is also included for an apparatus for the process.

Description

The invention relates to a method for growing and / or treating cells according to that in the preamble of claim 1 defined in more detail. The invention also relates to a device for breeding and / or Treat cells.

In the older application of the inventor DE 199 35 643.2 is a method and an apparatus of the beginning described type.

It has now been found that the formation of a Cell layer and cell growth significantly improved  is when you pressurize the cells out puts. For this it is already known from practice a cell culture room through mechanical devices, such as B. a stamp, e.g. B. as in US 6,060,306, to charge. In addition to the necessary con structural effort corresponds to such a burden due to the heterogeneity achieved in vivo conditions. In US 5,928,945 at all namely about shear stress using a culture medium mechanical load of z. B. Cartilage cells tried. However, this is unphysiological, since e.g. B. in articulated such perfusions are not sufficient. to In addition, the oxygen content is due to the limi not transport capacity for oxygen set all tissue types in physiological ranges bar (McLimmans et al. 1968).

An apparatus is also described in US Pat. No. 6,060,306 in which a cartilage construct within a culture chamber by moving the outer walls as in an egg a bellows is moved. These movement processes ha ben the disadvantage that the movement pattern a high cause mechanical stress on the membrane structures,  especially if this is due to permeability constraints conditions for oxygen are particularly thin, d. H. a dic ke in the micrometer range. This leads to the membrane tear after a few days and the products become non-sterile and therefore not for implantations are more suitable. Furthermore, the membrane can be opened because of the movement patterns that are constantly convex-concave Cause deformation, even just punk accordingly generate pressure deformations. This is what happens Oscillations in the culture medium area and pressure hetero biological tissue in the bioreactor.

The task at hand therefore lies with the task reasons, a method and an apparatus for breeding and / or treating cells, whereby so as far as possible in-vivo conditions are simulated can.

In particular, it should be possible to produce completely homogeneous pressure conditions in all areas within the bio-actuator tissue within the bio-actuator, without any convex-concave membrane deformations of the outer walls of the system. If these membranes are also to perform oxygenation tasks in order to avoid undesirable heterogeneities in the transport distances for the O ₂ diffusion and thus deficiency states, then these must be at a stable and as short a distance as possible from the biological constructs and ideally always be in direct contact with them. that is, they have to follow all movements of the biological construct in an identical manner instead of inducing it like a bellows.

According to the invention, this object is achieved by the drawing part of claim 1 called ge solves.

A device for solving the problem is apparent from claim 3.

In vivo conditions can be simulated very well by a gas medium or by the formation of a gas space. So it is z. B. possible to expose the cell layer in the cell culture space very specifically to changing and homogeneous pressure loads, which can be controlled very often and as quickly as desired without great effort. As soon as pressure is exerted on the outer film layer via the external gas phase, the entire film layer is placed around the implant. This means that even very complex 3-D structures can be completely encased. The diffusion paths for O ₂ are thereby reduced to almost 0 at the same time. With 3-D structures, the foils / membrane layers can ideally already have appropriate shapes for the implant, so that a simple modeling is possible in the event of pressure loading.

The formation of a gas space can be done in different ways se done. For this, e.g. B. a pressure-resistant housing be provided, which at the same time advantageously is designed as a bioreactor. In such a way The cell layer in the cell can then act as a bioreactor Kulturraum are treated as an independent unit. For this purpose, the unit made of the microporous film, the cell culture room and a protective film be that also surrounds the support structure. In this If necessary, the unit can then be removed from the Remove bioreactor - if necessary after removal connections for oxygen and / or nutrient medium - and Z. B. freeze or transport as an implant.  

Advantageous further developments and refinements ben from the following based on the drawing embodiments described in principle.

It shows:

Figure 1 is a schematic representation of a first embodiment with articular cartilage, which are grown on a support structure layer, which is intended to represent a knee joint. and

Fig. 2 is a schematic diagram of a device, similar to that of FIG. 1, with a counter structure.

The invention is based on a Vorrich to form hyaline cartilage than to grow described the cells for a knee joint. Selbstver this type of application is only for example to see. There are still numerous within the scope of the invention other applications possible.  

On a support structure 1 formed from tricalcium phosphate, cartilage cells are placed as cells 2 to form a cell layer. As can be seen, the support structure 1 corresponds in its shape to a knee joint. A microporous film 3 is placed over the cells 2 , which must be sufficiently elastic so as not to hinder cell growth. As a material for this. B. PTFE possible. The microporous film 3 must either seal the support structure 1 tightly or enclose it completely. In the illustrated embodiment, it lies laterally against the support structure 1 , while the support structure 1 rests on a plate 4 . The microporous film 3 is hen with an inlet connection 5 and a return connection 6 verses, can be brought about the nutrient medium in a continuous process. At the same time, oxygen can also be supplied here, provided that oxygen is not introduced from the environment around the microporous film 3 through the film itself.

As can be seen, the microporous film 3 forms, together with the support structure 1, a cell culture space 7 for the cells 2 arranged therein.

The microporous film 3 and the support structure 1 bil - possibly together with the plate 4 - a unit which is inserted into a pressure-tight housing 8 . The pressure-tight housing 8 forms a bio reactor. A connection to a pressure medium 10 is created in the pressure-tight housing 8 via a pressure connection 9 . In this way, a gas space 11 is created in the pressure-tight housing 8 , which is placed under changing pressure loads by the pressure medium 10 in the desired manner. These changing pressure loads have an effect on the cells in the cell culture space 7 via the microporous film 3 . The microporous film can also be omitted, so that the cells are directly modeled by the protective film. If a separate oxygen or air supply is provided for the cells 2 , any other gas can also be used as the gas medium.

If necessary, the unit formed from the microporous film 3 and the carrier structure 1 can also be surrounded by a further protective film 12 (see dashed line). The protective film 12 serves to transport the unit out of the bioreactor 8 and thus closes the unit sterile. Various foils can be used as protective foil 12 . Since a gas space 11 is formed in the interior of the housing 8 , the protective film 12 should accordingly be modelable and gas permeable. The gas space 11 can be formed both inside the protective film 12 and outside of it. In the latter case, a connection for egg ne connection to the pressure source 10 is therefore to be provided in the protective film 12 .

Instead of tricalcium phosphate for the support structure 1 , collagens can also be used in the bone replacement area. B. a meniscus can also be grown. Connective tissue structures, poly mers, such as polylactides or other chemical structures, can also be used.

It is essential that you can model shapes that correspond to a desired implant. In this way you can e.g. B. in the event of a defect, look for an implant of a certain size, for which purpose. B. via com putertomographic images, a corresponding analysis is carried out and then in the bioreactor 8 or, if appropriate, a desired support structure 1 is formed beforehand, on which the associated cells are then grown.

In addition, electrical connections can also be provided in the bioreactor 8 , through which one can apply electrical impulses to the cells 2 via corresponding connecting lines (not shown), with which even better in vivo simulations can be achieved.

The embodiment shown in FIG. 2 corresponds essentially to the form discussed in FIG. 1, which is why the same reference numerals have been retained for the same parts.

The only difference is that a meniscus structure 13 has been applied over the microporous film 3 , over which in turn a support structure 14 is located. In this way, even better in-vivo conditions are simulated. To form a transporta ble unit, the whole can then also be surrounded by a protective film 12 through which connections 5 and 6 are guided for the supply and removal of nutrient medium and / or oxygen. The gas space 11 can in turn be formed outside of the protective film 12 or inside via a corresponding pressure connection.

As mentioned, the cells are introduced to form a cell layer in a cell culture space 7 between two foils or between a foil and the modelable carrier structure 14 . Likewise, the support structure can be attached bilaterally as in a mold with two or more halves. The shape can also be re-modeled during the settlement process, ie it can be adapted assuming a new shape.

It is essential that these contain cells / matrix fillable structure can be introduced into a chamber can by means of a gas or liquid phase a pressure load that is also cyclical in nature can be exposed. Instead of a gas medi Of course, a liquid medium can also be used be used.

The shape of the foils is fully adaptable to reversible pressure loading processes, as it is largely not subject to its own pre-tension. This means that what is important is not the elasticity of the foils, but the position-conforming contact of these foils, which is made possible by a very high level of compliance or stretch. The foils can thus be modeled or are maximally and homogeneously modeled by applying pressure. The foils can also be shaped in a profiling manner. Interior profiles for cell culture room 7 are also possible.

This outer film 12 can thus lie on the carrier substrate 1 to be settled as completely as possible initially, ie before the start of the pressure loading process, and can thus prevent wear and tear that is otherwise caused by a reversible pressure loading and also directly have a pro-filing effect. This profile can also be supported by the fact that the externally applied pressure has a shaping effect via a pressure profile.

In the treatment room, the substrate to be populated substrate 1 , z. B. in an in vivo-like form in the sense of a profile, structure also introduced as a hollow body (shell). These structures can then be colonized inside and / or outside as well as transmurally with substrates and cells. They can also be traversed with culture medium and oxygen.

The flexibility of the device is also advantageous and the process, since the inner bioreactor construction le are removable and therefore for transport and cry processes are accessible.

Claims (10)

1. Apparatus method for growing and / or treating cells on a modelable carrier structure, the cells for forming a cell layer in a cell culture space between the carrier structure or a film applied to the carrier structure and a second film, in particular one microporous film who introduced the, wherein oxygen and / or nutrient medium is introduced into the cell culture space, characterized in that the cell culture space ( 7 ) is exposed to changing pressure loads by a gas medium. 2. The method according to claim 1, characterized in that to the cell culture chamber (7) and at least partially around the support structure (1) a protective sheet (12) wherein a shape and composition is used as a support structure (1) is placed, which on the one in -vivo structure is largely simulated or reproducible, and that the two foils ( 3 , 12 ) with the support structure ( 1 ) are introduced as an independent unit in a bioreactor. 3. Device for growing and / or treating cells on a support structure, the cells for forming a cell layer being introduced into a cell culture space between the support structure or a film applied to the support structure and / or a microporous film, oxygen and / or Nutrient medium can be introduced into the cell culture space, characterized in that the cell culture space ( 7 ) is arranged in a gas space ( 11 ) which is provided with at least one pressure connection for connection to a pressure source ( 10 ) or which is provided by a pressure source ( 10 ). can be placed under changing pressure. 4. The device according to claim 3, characterized in that the gas space ( 11 ) is formed by a pressure-tight housing ( 8 ) surrounding the cell culture space ( 7 ) and the support structure ( 1 ). 5. The device according to claim 4, characterized in that the pressure-tight housing ( 8 ) bil det bil, in which the cell culture chamber ( 7 ) vorzugswei se between the microporous film ( 3 ) and a protective film ( 12 ) on and around the Support structure ( 1 ) is placed, is formed, the two foils ( 3 , 12 ) and the support structure ( 1 ) being arranged as a self-contained unit in the bioreactor. 6. Device according to one of claims 3 to 5, characterized in that the carrier structure ( 1 ) in the form and composition is at least largely formed according to an in vivo structure or is modelable. 7. Device according to one of claims 3 to 6, characterized in that the carrier structure ( 1 ) made of calcium material, in particular special calcium phosphate, is formed. 8. Device according to one of claims 3 to 6, characterized in that the carrier structure ( 1 ) is formed from collagen / extracellular matrix. 9. Device according to one of claims 3 to 6, characterized in that the carrier structure ( 1 ) made of a biodegradable polymer, such as. B. polylactide is formed. 10. Device according to one of claims 3 to 9, characterized in that a second structure ( 14 ) is arranged above the microporous film ( 3 ) or the support structure or the first outer film, which in form and optionally in its composition one in vivo counter-structure to the support structure ( 1 ) is formed after.