METHOD FOR PRODUCING TRANSPLANTABLE ORAL MUCOSA TISSUE

Abstract

The present invention relates to a method for producing transplantable oral mucosa tissue by selecting and choosing from oral mucosa biopsy, and to the use thereof as pharmaceutical composition, medicament or transplant or graft material, in particular for urethral reconstruction or cornea implantation. In particular, the invention relates to markers for identifying suitable transplantable oral mucosa tissue for the production of transplantable oral mucosa tissue, in particular for producing an autologous oral mucosa graft or tissue.

Claims

1. A method for selecting oral mucosa tissue for production of transplantable oral mucosa tissue, wherein cells are cultured on a surface of a support, wherein at least one potency marker is selected from at least one vascular endothelial growth factor (VEGF) having a threshold value of more than or equal to 10 pg/ml in supernatant after 24 h of seeding on the surface and at least one purity marker is selected from at least one acidic and basic (Type I and II) subfamilies of cytokeratins, wherein the amount of epithelial cells is more than or equal to 70%, optionally 80%, and/or at least one impurity marker is selected from at least one Cluster Differentiation 90, wherein the amount of non-epithelial cells is less than or equal to 30%, optionally 20%, wherein the markers are determined.

2. The method for selecting oral mucosa tissue according to claim 1, wherein the purity marker is selected from at least one acidic and basic (Type I and II) subfamilies of cytokeratins, wherein the amount of epithelial cells is more than or equal to 95%, and/or the impurity marker is selected from at least one Cluster Differentiation 90, wherein the amount of non-epithelial cells is less than or equal to 5%.

3. The method for selecting oral mucosa tissue according to claim 1, wherein first, the oral mucosa cells derived from a tissue biopsy are multiplied in a culture, and, second the cells are cultured on a surface of a support.

4. The method for selecting oral mucosa tissue according to claim 1, wherein the cells are cultured on the surface of a support having a density of at least 1.010E5 to 1.610E5 cells, optionally at least 1.310E5 cells/cm.sup.2.

5. The method for selecting oral mucosa tissue according to claim 1, wherein the proportion of fibroblasts in the isolated oral mucosa tissue in relation to the total cell number is less than or equal to 20%, optionally less than or equal to 5%.

6. The method for selecting oral mucosa tissue according to claim 1, wherein at least one potency marker is selected from at least one vascular endothelial growth factor (VEGF) having a threshold value of more than or equal to 10 pg/ml in the supernatant after 48 h of seeding on the surface.

7. The method for selecting oral mucosa tissue according to claim 1, wherein the support comprises a dissolvable membrane consisting of one or more biodegradable polymers, optionally biocompatible polymers or water-soluble polymers.

8. The method for selecting oral mucosa tissue according to claim 7, wherein the support comprises biodegradable polymers selected from the group of biological or non-biological origin, alpha-hydroxy acids, lactic acid and/or glycolic acid, polylactides, polyglycolides, polylactide-co-glycolides, and copolymers with polyethylene glycol, polyanhydrides, poly(ortho)esters, polyurethanes, Polyglycolic acids, Polylactic acids, Polycaprolactone, polybutyric acids, polyvaleric acids, polylactide-co-caprolactones, polyvinyl pyrrolidone (PVP), glycans, glycosaminoglycans and polysaccharides, such as pullulan, hyaluronan, gellan, cellulose, chitosan, alginate, and collagen.

9. The method for selecting oral mucosa tissue according to claim 1, for producing an autologous oral mucosa graft or tissue.

10. Isolated oral mucosa tissue obtainable by a method according to claim 1.

11. Isolated oral mucosa tissue obtainable by the method according to claim 10, wherein a proportion of fibroblasts in the isolated oral mucosa tissue in relation to the total cell number is less than or equal to 5%.

12. Isolated oral mucosa tissue obtainable by the method according to claim 10, wherein the proportion of lymphocytes and adipocytes in the isolated oral mucosa tissue in relation to the total cell number is less than or equal to 1%.

13. A graft or transplant comprising oral mucosa obtainable by the method according to claim 9 or isolated oral mucosa tissue thereof.

14. The graft or transplant comprising oral mucosa tissue according to claim 13 for treatment of one or more oral mucosa defects and/or diseases selected from vascular disorders, including diabetic foot, ulcer due to atherosclerosis, as well as diseases which require additional tissue for reconstruction, optionally skin burns, shrunken bladder, bladder dystrophy, small bladder after excision of bladder tumors, esophageal atresia, missing tissue after excision of esophagus tumor, ureteral stricture, ureteral tumor, urethra diseases, optionally urethral stricture, urethra malformation, hypospadias, corneal diseases, optionally limbal stem cell deficiency, cornea injury, and optionally having a scaffold or matrix.

15. The graft or transplant comprising oral mucosa tissue according to claim 13, wherein the oral mucosa tissue is fixed on the support, wherein the support is a dissolvable, self-dissolvable membrane or a biodegradable membrane.

16. An applicator comprising the graft or transplant according to claim 12.

Description

EXAMPLE 1

[0061] In order to qualify VEGF as a parameter for potency testing according to the invention, the inventors have performed a dosing study using different cell seeding densities and using an ELISA method for VEGF quantification.

[0062] Cultured oral mucosa cells that were isolated from an oral mucosa biopsy of an adult human donor were used for this study. Biopsy taking and processing as well as cell culturing were performed according to standard protocols. The cells of the final cell suspension were characterized by immunocytochemical staining using antibodies against cytokeratin as a marker for epithelial cells and against CD90 as a marker for other cell types like fibroblasts to exclude substantial contamination by non-epithelial cells. Several biodegradable matrices were seeded with increasing cell numbers of the finally harvested cell suspension. For each applied cell density membranes were prepared in triplicates. Each three membranes were seeded with six different cell densities from 0.0410E625% cells/cm.sup.2 to 0.1910E625% cells/cm.sup.2.

[0063] The used ELISA kit for quantitative VEGF determination was intended for samples with human VEGF concentrations up to 6,000 pg/ml. A linear correlation up to this concentration was found using the standards provided with the test kit. The corresponding calibration curve together with its equation is provided in FIG. 1. The FIG. 1 also shows the result of VEGF determination in the samples taken after 24 h and 48 h from the supernatant of the cell seeded membranes. The concentrations of VEGF found after 48 h incubation were higher than those found after 24 h incubation. Paired t-test analysis revealed a highly significant difference between the VEGF concentrations found after 24 h and 48 h incubation (p<0.0001).

[0064] The following results were found regarding the effect of cell seeding density and the dependent release of VEGF: After 24 h incubation statistically significant differences of VEGF concentrations were found in medium samples from membranes seeded with 0.04 vs. 0.0710E625% cells/cm.sup.2, 0.07 vs. 0.1010E6 cells/cm.sup.225% and 0.10 vs. 0.1310E625% cells/cm.sup.2. No significant difference was found for the VEGF concentrations in medium samples taken from degradable matrices seeded with 0.13 vs. 0.1610E625% cells/cm.sup.2 or 0.16 vs. 0.1910E625% cells/cm.sup.2, respectively.

[0065] Analysis of the medium samples taken 48 h after incubation showed a slightly different result. No significant difference was found for the difference in VEGF concentration between medium samples taken from degradable matrices seeded with 0.04 vs. 0.0710E625% cells/cm.sup.2. Significant differences regarding VEGF concentration were found between medium samples taken from membranes seeded with 0.07 vs. 0.1010E625% cells/cm.sup.2, as well as 0.10 vs. 0.1310E625% cells/cm.sup.2. The difference between the VEGF concentration in medium samples taken from matrices seeded with 0.13 vs. 0.1610E625% cells/cm.sup.2 was not significant.

[0066] However, the difference in VEGF concentration between medium samples taken from matrices seeded with 0.16 vs. 0.1910E625% cells/cm.sup.2 was significant.

[0067] Overall, a density-dependent increase of VEGF concentrations was found for cell seeding densities up to 0.1010E625% cells/cm.sup.2 but not or to a lower extent for higher cell seeding densities. Maximum VEGF concentrations were already reached at a cell seeding density of 0.1310E625% cells per cm.sup.2 in accordance with the present invention.

EXAMPLE 2

[0068] ELISA: To determine the concentration of VEGF as a parameter for potency testing 1 ml of the supernatant culture media from cultured cells on biodegradable matrices are taken off and determined per ELISA.

[0069] The VEGF measurement is based on a commercial ELISA kit (R&D Quantikine Elisa for human VEGF, catalog #DVE00/SVE00/PDVE00).

[0070] To conduct the assay, aliquots of the samples to be tested and of the VEGF standards are added in triplicate to the wells of the anti-human VEGF coated microwell plate. The plate is then incubated 2.5 h at room temperature. After washing four times with washing buffer, the prepared biotinylated detection antibody is added to the wells. The antibody is incubated one hour at room temperature followed by four times washing and the addition of the prepared streptavidin solution. After incubation for 45 minutes at room temperature, the plate is washed four times and the TMB substrate reagent is added to the wells. After 15 minutes incubation the reaction is stopped by adding the stop solution to the wells. The absorbance at 450 nm is measured by means of a microplate reader immediately after addition of the stop solution. Concentrations of VEGF are calculated using a calibration curve. As positive control, endothelial cell specified culture medium of PromoCell is used. As negative control, the same medium without supplements is used.

[0071] Angiogenesis-Tube Formation Assay:

[0072] It is known that angiogenesis is characterized by several cellular events including endothelial cell migration, invasion and differentiation into capillaries. In vitro endothelial tube formation assays are used as a model for studying endothelial differentiation and modulation of endothelial tube formation by antiangiogenic agents. Image acquisition and quantification of fluorescently labelled cells are achieved using appropriate software. In our case, human endothelial stem cells are seeded on 6-well plate and attached for 48 h in growth medium and differentiation of the cells using differentiation medium. Afterwards, endothelial cells are seeded onto Corning Matrigel Matrix where they are feeded with conditioned medium taken from oral mucosa cell cultures on biodegradable membranes. Due to the presence of VEGF in the conditioned medium, endothelial cells are induced to form angiogenic tubes in the 3D Matrigel. Angiogenic potency is determined by software assisted quantitative assessment of microvessel formation (Carpentier, G. Angiogenesis Analyzer for ImageJ. ImageJ News 9 Nov. 2012).

[0073] For the investigation of potency of cultured oral mucosa cell on biodegradable matrices, The conditioned culture media from several batches were tested in both ELISA and angiogenic tube formation assays.

[0074] The results of the VEGF ELISA assay are shown in Table 4.

TABLE-US-00001 TABLE 4 VEGF concentrations in tested culture media. All samples show a value of more than 10 pg/ml, except No. 3 and 4, which were much lower. The two latter conditioned media (2 and 3) were thermally pretreated, resulting in the denaturation of VEGF. Sample No. VEGF [pg/ml] STDV [pg/ml] 1 644.9 4.9 2 194.4 1.8 3 1.9 0.2 4 2.7 0.2 5 432.0 5.7 6 559.5 2.3 7 528.9 2.5 8 425.8 5.7

[0075] The same culture media were used in the tube formation assay. Results are shown in FIG. 3 and FIG. 4.

[0076] These results indicate that the culture media conditioned by the cells seeded on the biodegradable membranes exert an angiogenetic effect on the HUVEC cells, comparable to the highly specific and inducing angiogenetic endothelial medium from PromoCell which was used as a positive control. Generally, it has to be noted that despite the fact that glucose, insulin and other substances are already consumed by cultured oral mucosa cells in media after two days of incubation on the membrane, the effect is comparable to the positive control. Interestingly, two media samples (No. 3 and 4) did not meet the threshold value set regarding VEGF concentration (10 pg/ml) in accordance with the invention. The same two media showed no microscopic detectable tube formation (FIG. 3) and no branching (FIG. 4) as well. These two media were thermally treated prior the ELISA and angiogenic assay, resulting in the denaturation of VEGF.

[0077] The following table (Table 5) summarizes the data obtained for the stated samples for ELISA and Angiogenesis assay. The correlation efficient as well as the corresponding p-values were calculated based on these results and are also indicated in the table.

TABLE-US-00002 TABLE 5 Calculation of the correlation of VEGF ELISA and Angiogenesis assay. ELISA Angiogenesis VEGF Nr. of Nr. of Total branching Sample ID [pg/ml] Segments meshes length [m] 1 644.9 219.83 45.75 20434.71 2 194.4 174.08 30.42 18765.30 3 1.90 0.62 0.38 286.48 4 2.70 7.20 1.40 176.74 5 432 128.43 20.71 15350.65 6 559.5 169.50 30.44 19436.22 7 528.9 140.95 23.27 16758.92 8 425.8 151.00 23.00 17866.69 Correlation 0.997 0.980 0.985 p-value .sup.0.001 (**) .sup.0.007 (**) .sup.0.005 (**)

[0078] To assess a potential correlation of VEGF ELISA data with angiogenesis measurements, angiogenesis values (nr. of segments, nr. of meshes, and total branching length) were compared to the corresponding VEGF concentrations. Interestingly, all values showed a significant correlation between ELISA and angiogenesis measurements. These results indicate that VEGF ELISA can be used as a marker assay for the potency measurements of the oral mucosa cells according to the invention.

EXAMPLE 3

[0079] Phenotypic characterization of oral mucosal cells, Keratinocytes-via flow cytometry (FACS) Investigation of cell identity (epithelial cells 70%) and purity (Fibroblasts 30%)

[0080] The inventors conducted a phenotypic characterization of in vitro expanded oral mucosal cells (225 days of culture) encompassing 100 independent batches. The purpose of this study was the confirmation of identity of cultivated human oral mucosal cells using flow cytometry. Pan-cytokeratin antibody was used to identify epithelial cells (analysis of cell identity). Cytokeratins, a group comprising at least 29 different proteins, are characteristic of epithelial cells.

[0081] The used pan-cytokeratin antibody was a cocktail of two mouse monoclonal antibodies (AE1/AE3) (supra).

[0082] For detection on impurities, CD90 staining was used for all 100 patients for the detection of any existing fibroblasts.

[0083] For each analysis approx. 500.000 cells were fixed with 1 mL 0.01% formalin and post-fixed with ice-cold ethanol. The fixed cells were then permeabilized with 0.1% Triton/1% BSA in PBS, and aliquots of the permeabilized cells were stained with a 1:500 dilution of the AE1/AE3 or CD90 antibody (Dako, M3515) in 1% BSA or with the respective buffer control. The cells were differentiated with an Alexa Fluor 488-coupled goat-anti-mouse IgG and resuspended in 1% BSA for FACS analysis (one-colour histograms).

[0084] FACS analysis and evaluation of data was performed using a Becton Dickinson FACS Calibur device and the CellQuest Pro software package. FIG. 6 shows an example of FACS results for cytokeratin, obtained for the first analysed batch in this series of experiments.

[0085] Detection of fibroblastic impurities and confirmation of epithelial identity in oral mucosal cells via immunocytochemistry:

[0086] To confirm the results of FACS analysis, immunofluorescence staining was additionally carried out with a keratinocyte specific marker (Pan-Cytokeratin) for identity and CD90 antibody for quantifying potential cellular impurities in terms of fibroblasts

[0087] Applied method of cell staining with pan-cytokeratin and CD 90 for immunocytochemical analysis determination of purity and Identity of cells: [0088] Wash the cells 31 min with PBS solution [0089] Remove PBS. [0090] Fix the cells by adding 4% PFA or acetone for 10 min at RT. [0091] Remove 4% PFA or acetone solution [0092] Wash the cells 31 min with PBS solution [0093] Incubate the cells with 100 l antibody (CD90 with concentration 1:50 and PCK with concentration 1:100 at the same time) solution in each well at RT for 4 hrs. in the dark [0094] Incubated the cells with 100 l of Hoechst staining solution for 3 min at RT.

[0095] The results of immune-cytochemical analysis in immunofluorescence microscopy is in accordance with the results of FACS confirming the epithelial identity of cells and a fibroblastic impurity of less than 5% (cf. FIG. 6).

[0096] FIG. 1: Quantitative determination of VEGF concentrations. The figure A shows the results for samples with known VEGF concentration and the measured absorbance. The corresponding calibration curve and the equation are indicated in the picture. The figure B depicts the calculated means and standard deviations of measured VEGF concentrations of supernatant culture medium taken from membranes seeded with increasing cell densities (indicated on the x-axis). Culture medium was taken after either 24 h (left columns) or 48 h (right columns), respectively.

[0097] FIG. 2: Correlation between VEGF release and cell coverage of the membrane. The VEGF concentrations of the indicated samples were measured after either 24 h (figures on the left side) or 48 h (figures on the right side). A positive correlation was found in both cases. The corresponding correlation coefficient (p) for each calculated linear correlation is indicated.

[0098] FIG. 3: Tube formation assay with culture medium from 8 batches. Human Umbilical Vein Endothelial Cells (HUVEC) were cultivated until proper number in Endothelial Cell Growth Medium (PromCell GmbH). HUVEC cells were detached from the cell culture flasks using Trypsin/EDTA into single cell suspension. Corning Matrigel was carefully thawed and coated in the bottom of 48-well plate. 150 l cell suspension with 810.sup.4 cells were seeded unto the Matrigel surface. After 8 hours, HUVEC formed angiogenic tubes, and were stained with Calcein AM (Live Cells, Green) and Propidium Iodide (Dead Cells, Red). Fluorescence Photos were taken in the channel of Green, Red and Bright Field. Batches from left to right, from top to bottom: 1, 2, 3, 4, 5, 6, 7, 8. Last picture: HUVEC fed with PromoCell Endothelial Cell Growth Medium as positive control (9).

[0099] FIG. 4: Quantification of tube formation of 8 batches. Tube formation as described and illustrated in FIG. 3 was quantified for the analyzed samples using the software program ImageJ.

[0100] FIG. 5: Example of phenotype analysis via flow cytometry for oral mucosa cells. The example shows results for the first analysed sample (human MSH 02.03.10 (Charge 1)).

[0101] FIG. 6: Compilation of all determined percentages of pan-cytokeratin expression (Confirmation of cell identity). FIG. 7 presents the results of 100 independent batches of oral mucosal cells regarding the expression of pan-cytokeratin. One hundred independent patient charges were analyzed and for each batch The mean percentage of pan-cytokeratin expression in all 100 analyzed cultures was 98.2%.

[0102] FIG. 7: Compilation of all determined percentage CD90 values (Confirmation of cell purity.)