EP2809369A1

EP2809369A1 - Matrix for cell colonization

Info

Publication number: EP2809369A1
Application number: EP13706421.8A
Authority: EP
Inventors: Martin GÖRNE
Original assignee: Bioenergy Capital AG
Current assignee: Bioenergy Capital AG
Priority date: 2012-02-01
Filing date: 2013-02-01
Publication date: 2014-12-10
Also published as: CN104245003A; WO2013113516A1; RU2014135283A; EP2623135A1

Abstract

The invention relates to a cell implant matrix which has a connective porosity of over 80% and which consists predominantly of a mixture of bioresorbable polymers. The matrix has a disk shape, and a surface layer on one face of the disk has less than 20% of the average pore density of the other faces. The matrix is produced by providing a bioresorbable polymer layer; stacking a mixture of a water-soluble solid, at least two polymers which differ with respect to resorption rates, and a solvent for one of the polymers onto the polymer layer; evaporating the solvent and subsequently optionally compacting the mixture; and watering the compact body in order to remove the salt.

Description

Matrix for cell colonization

The present application relates to porous matrixes for cell colonization for therapeutic or diagnostic purposes.

Cell implants based on porous matrices of biocompatible polymers are known from WO 2004/108810 AI. In such matrices, the pores are cross-linked and serve as a template for the location of cells in vivo (eg, therapeutically) or in vitro (eg, diagnostically). In transplantations, such a bioresorbable matrix can serve for the temporary localization of the graft.

The known templates are not yet fully satisfactory in some applications, in particular with regard to the clinical results.

The invention therefore sets itself the goal of achieving an improvement in the clinical performance of the template.

To this end, the invention proposes to provide on one side of the usually disc-shaped template a surface with less than 20% of the average pore content of the other side (s). This asymmetric structure allows the template to be placed in the body so that the implanted vital cells stay in it longer.

In a further aspect, the invention provides methods of making porous bioresorbable matrices, initially forming a polymer layer without a pore former, followed by coating a mixture of at least two polymers, a solvent for one of the polymers, and a water-soluble pore former, followed by evaporation of the solvent and then water to remove the pore-forming agent. Between these last two steps, in one variant, pressurization for compacting may still take place. Both methods lead to highly porous polymer matrix disks, but one side of which has a pore-free or at least low-pore membrane. Despite this membrane, the supply of the cells in use in the case of the application is sufficient, the However, loss rates due to emigration are considerably reduced. If, in one variant, such a template serves as connective tissue support, the continuous polymer layer can have sufficient hold for z. B. Provide suture for fixation of the template.

Further features of the invention will become apparent from the following description of exemplary embodiments in conjunction with the claims and the figures. The invention is not limited to the described embodiments, but determined by the scope of the appended claims. In particular, the individual features in embodiments according to the invention can be realized in a different number and combination than in the examples given below. In the following explanation of an embodiment, reference is made to the accompanying figure, which shows a flow chart for a method according to the invention. In one main application, matrices are provided for colonization with functional cells, for example hepatocytes and / or Langerhans' islet cells. Such biochemical function cells attach to the inner walls of the pores of the foamy matrix (attachment rates over 80% or, suitably coated, over 95%) and can be transplanted with the matrix into mesothelial pockets, ideally the cell donor itself In this case, no rejection reaction takes place, but only a comparatively mild, for the therapeutic process favorable foreign body stimulus is exercised. Within a few weeks, the matrix becomes vascularized and the implanted cells no longer rely on diffusive supply. The matrices are arranged so that the pore-poor (or -free) side is on the inside and the pore-rich side on the outside to keep the loss rate low due to the migration of the cells. In parallel, a gradual dissolution of part of the degradable matrix occurs (within 3-4 months, or at least 2 and / or less than 7 months), thereby affecting the physiological milieu in a manner which is also conducive to therapeutic success. It is useful if part of the Polymer blend eroded more slowly (ratio of degradation times at least 5) and the structural cohesion longer time, z. B. 2.5-3 years (or at least 2 and / or less than 5 years) guaranteed. Such polymers are useful based on α-hydroxycarboxylic acids such as lactic acid and / or glycolic acid, eg. PLA or PLGA. Manufacturers of such polymers approved for use in the human body indicate the nominal degradation times that are relevant here. First, a solution of one of the polymers used in chloroform allowed for medical purposes is poured into a mold and the solvent is evaporated off at 45 ° -65 °. Then, a polymer mixture of defined particle size distribution is mixed with a saline granules also defined particle size distribution, mixed with a solution of one of the polymers in chloroform and then added to the already prepared polymer layer. For this preform, the solvent evaporates at slightly elevated temperature (45 ° C-65 ° C) and this can then be compacted if desired by applying pressure. Subsequently, the compact is watered to provide the desired porosity by removing the salt. However, the initially produced polymer layer remains pore-free. Depending on the application, the thickness of the low-pore layer can be adjusted by the amount and concentration of the initial solution. For example, a very thin membrane is obtained when the concentration of the solution is low (eg 4% in chloroform, slowly degradable polymer) and the fill level is small (eg 0.1-1 mm, for example 0.3 mm). If a more mechanically robust structure is desired, higher (eg 5-50 mm, typically 20-25 mm) can be introduced at the same concentration. The evaporation of the chloroform then takes longer (1.5 h). In the first case, the resulting membrane has a thickness of about 10-20 pm, in the latter case about 0.5-1 mm.

The saline particles of the mixture to be coated are somewhat coarser (median at 350-370 pm) than the polymer particles (median of the slower degradable polymer between 210 pm and 230 pm, that of the faster degradable polymer between 150 pm and 170 pm). The distribution widths (5% / 95%) are similar, namely about ± 85-95 μπι for salt or polymer in total. The distribution form can be bi- or trimodal. The composition of the layer mixture is about 96% salt, 1-1.5% solid polymer and another about 3-5% dissolved polymer, wherein the volume fractions of solids and liquid are about the same. Overall, the proportion of the rapidly degradable polymer is only about 5-20% of the polymer. The total thickness of the pore-forming layer is 5-6 mm. In the variant of a more stable initial layer, the salt can be chosen a little coarser (median 400-420 m). In this case, the total thickness of the pore-forming layer 4-5 mm and can be dispensed with the compaction with pressure. The water lasts about 24 h and is followed by drying at 45-50 ° C. If a coating is applied, the matrix lies with the low-pore side and is therefore predominantly coated on the open-pored side.

The polymers used here are z. Available from the company Evonik and bear the designations L210s, L210, L09s, L207s, L206s (slower degradable PLGA polymers) and RG502, RG502H, RG505 (faster degradable PLGA polymers).

When used outside the body, a matrix as described above may serve to fix cells exposed to agents in a bioreactor. For example, defined cell types may be screened for whether or not they respond to candidate drugs, and the therapy may be scheduled in response to assay results obtained in this manner. Similarly, drug development can be simplified ver ^¬ because toxicity is detected early.

Claims

claims

Cell implant matrix with a connective porosity of more than 80%, mainly consisting of a mixture of bioresorbable polymers, the matrix having a disc shape, characterized in that

the surface on one side of the disk has less than 20% of the average pore content of the other sides of the disk.

The matrix of claim 1, wherein the matrix consists predominantly of poly (hydroxy) carboxylic acids.

A matrix according to any one of the preceding claims, wherein absorption rates of two of the polymers forming the mixture, each accounting for at least 10% of the mixture, differ by a factor of more than 5.

4. Use of the matrix according to one of claims 1 to 3, for colonization with vital cells and for exposure of the vital

Cells a predetermined test agent outside the body.

Preparation of a cell-implant matrix of different porosity, comprising:

Producing a bioresorbable polymer layer;

laminating on one side a mixture of a water-soluble solid, at least two polymers having different rates of absorption and a water-immiscible solvent for at least one of the polymers on the polymer layer;

Evaporation of the solvent; and

Watering the agglomerate to remove the water-soluble solid from the coated layer. 6. The method of claim 5, further comprising applying pressure to compact the mixture after evaporation of the solvent.

7. The method according to claim 5 or 6, wherein chloroform is used as the solvent. 8. The method according to any one of claims 5 to 7, wherein

Resorptionsraten two of the mixture forming polymers, each accounting for at least 10% of the mixture differ by a factor of more than 5.

9. The method according to any one of claims 5 to 8, wherein the thickness of the initially prepared layer is 0.002 to 2.5 mm. 10. The method of claim 9, wherein the thickness of the initially prepared layer is 0.005 to 0.025 mm.

The method of claim 9, wherein the thickness of the initially formed layer is 0.25 to 2.0 mm.

12. Use of the matrix prepared by the method according to any one of claims 5 to 11, for colonization with vital cells and for exposing the vital cells to a predetermined test agent outside the body.

Use of the matrix according to any one of claims 1 to 3, or the matrix prepared by the method according to any one of claims 5 to 11, in a therapeutic process for the treatment of the human body.