METHOD FOR PRODUCING A SKIN EQUIVALENT, AND USE THEREOF FOR IN VITRO TESTS AND IN VIVO TRANSPLANTS

Abstract

A method for producing a skin equivalent comprising the steps of: a) producing a dermal matrix containing a first layer and a second layer comprises the steps of i) providing the first layer; ii) providing a polymerizable solution comprising a liquid and at least one polymer; iii) adding the polymerizable solution to the first layer; iv) polymerizing the solution to form the second layer; v) compressing the second layer, wherein as a result of the compression the liquid content of the second layer is reduced and the first layer and second layer are joined together to form the dermal matrix; and vi) introducing at least one cavity into the dermal matrix; b) introducing at least one hair follicle and/or hair follicle germ into the at least one cavity of the dermal matrix produced in step a) to produce a skin surrogate; and c) adding at least one further cell population.

Claims

1. A method for producing a skin equivalent comprising the steps of: a) producing a dermal matrix containing a first layer and a second layer comprising the steps of: i) providing the first layer; ii) providing a polymerizable solution comprising a liquid and at least one polymer selected from collagen, elastin and/or hyaluron; iii) adding the polymerizable solution to the first layer; iv) polymerizing the solution to form the second layer; v) compressing the second layer, wherein as a result of the compression the liquid content of the second layer is reduced and the first layer and second layer are joined together to form the dermal matrix; and vi) introducing at least one cavity into the dermal matrix; b) introducing at least one hair follicle and/or hair follicle germ into the at least one cavity of the dermal matrix produced in step a) to produce a skin surrogate; and c) adding at least one further cell population selected from fibroblasts, endothelial cells, adipocytes, nerve cells, gland cells, melanocytes or keratinocytes to the skin surrogate to produce the skin equivalent.

2. The method according to claim 1, wherein the first layer comprises a natural skin substance or an artificial skin substance.

3. The method according to claim 1, wherein the first layer contains substantially no living cells.

4. The method according to claim 1, wherein the polymerizable solution additionally contains cells.

5. The method according to claim 1, wherein prior to step iv) the polymer in the polymerizable solution is patterned by agitation.

6. The method according claim 1, wherein compression of the second layer proceeds by centrifugation, sedimentation or mechanical application of force.

7. The method according to claim 1, wherein method steps iv), v) or vi) take place in succession and/or simultaneously.

8. The method according to claim 1, wherein introduction of the at least one cavity into the dermal matrix in step vi) proceeds during or after the polymerization in step iv), during the polymerization.

9. The method according to claim 1, wherein the at least one cavity is introduced into the dermal matrix by piercing, excision or by means of an impression mold.

10. The method according to claim 1, wherein the cavities have an average diameter of 0.001 mm to 5 mm.

11. The method according to claim, wherein the cavities have a depth of 0.05 mm to 10 mm.

12. The method according to claim 1, wherein one cavity or a plurality of or all of the cavities in the dermal matrix is/are coated with extracellular matrix proteins prior to introduction of the hair follicle and/or hair follicle germ in step b).

13. A skin equivalent produced using the method according to any one of claim 1.

14. A transplant comprising an effective amount of skin equivalent according to claim 13, optionally together with pharmaceutically acceptable adjuvants.

15. A method for in vitro screening of substances with skin- and/or hair-regulating, comprising the steps of: a) providing a sample of the skin equivalent according to claim 13; b) dividing the sample into portions; c) incubating at least one portion with substances which are to be tested; and d) comparing the parameters of the skin and/or hair in the portion with another portion which has not been incubated with the substances.

Description

FIGURES

[0101] FIG. 1 Photographic image of a dermal matrix.

[0102] FIG. 2 Photographic image of a skin equivalent, a hair follicle and hair follicle germs.

[0103] FIG. 3 Photographic image of an impression mold for introducing cavities into a dermal matrix.

[0104] FIG. 4 Schematic diagram of an impression mold for introducing cavities into a dermal matrix. A: sectional view, B: plan view.

[0105] FIG. 5 Schematic diagram of an impression mold for introducing cavities into a dermal matrix. A: plan view of the top of the impression mold, B: plan view of the bottom of the impression mold, C and D: side view.

EXAMPLES

[0106] Isolation of the Various Cell Types from Hair Follicles

[0107] DPF, CTSF, MC and KC were isolated from the human hair follicle in accordance with a modified form of the standard protocols (described for example in Mager) et al.). Isolated hair follicles from punch biopsies or dissected hair follicles from an FUT hair transplantation were immobilized on the hair shaft with a pair of forceps and the connective tissue sheath was incised and diametrically separated therefrom carefully with another pair of forceps, so everting the bulb and exposing the dermal papilla and the hair shaft with the hair matrix. In this manner, the proximal part of the bulb with the connective tissue sheath fibroblasts and the dermal papilla could very easily be separated from the remainder of the hair follicle with the assistance of a needle/cannula. The hair shaft, which contains the hair matrix keratinocytes and melanocytes which are likewise required, was also optimally dissected for further culturing.

[0108] The dermal papillae and connective tissue envelopes extracted in this manner from the hair follicles which were taken were each collected in a separate vessel comprising medium. The DPF and CTSF were dissolved out and isolated from the surrounding tissue by gentle tissue dissociation using a tissue dissociator and the associated extraction kit (for example gentleMACS Dissociator #130-093-235, whole skin dissociation kit #130-101-540, Miltenyi Biotec). To this end, the isolated tissue fragments (dermal papilla, connective tissue envelope and hair shaft with epithelial/neuroectodermal cells), 435 l buffer L, and an enzyme mix of 12.5 mg enzyme P and/or 4.50 mg enzyme D and/or 2.5 mg enzyme A were each introduced into a gentleMACS C tube and carefully mixed.

[0109] The sample was incubated in a water bath at 37 C. for 1-3 hours or overnight, longer incubation times increasing cell yield. After incubation, the sample was diluted by adding 0.5 ml cold cell culture medium. The C tube was sealed and fastened upside down onto the sleeve of the gentleMACS dissociator. The h_skin_01 program was then run. Once the program was complete, a short centrifugation step was then carried out to collect the test material on the bottom. The cells could be washed with fresh medium and the cell suspension separated by a 70 m filter. It is possible to dispense with the above-described addition of enzymes, which are undesirable in the case of direct autologous therapy, with the assistance of further dissociation runs, although in this case lower cell yields must be expected.

Production of Cells from a Stromal Vascular Fraction (SVF)

[0110] A 1 liter fraction from tumescent liposuction of subcutaneous abdominal or hip fat was taken and prepared for example using the established PureGraft method (Cytori GmbH, Switzerland). This involved centrifugation and concentration in order to remove the tumescent solution. Digestion was then performed for 60 min at 37 C. in 0.15% (w/v) collagenase NB 6 GMP grade from Clostridium histolyticum (0.12 U/mg collagenase; SERVA Electrophoresis GmbH) diluted in phosphate-buffered saline (PBS; Gibco). After centrifugation at 180 g for 10 minutes, the lipid-rich layer was discarded and the cell pellet washed once with PBS. Erythrocytes were then dissolved by 2 minutes' incubation in lysis buffer (0.15 M ammonium chloride, Sigma-Aldrich). The stromal vascular fraction (SVF) obtained was suspended in complete medium (CM, Gibco).

Production of a Skin Equivalent

[0111] The dermis equivalent was built up on the basis of a 2 mm thick collagen/elastin matrix (Matriderm, Dr. Suwelack Skin & Health Care AG). A piece was punched out using a round punch and transferred into a Transwell. This was coated on ice with a collagen I solution containing dermal fibroblasts. The silicone plunger with the cylinders was gently rotated. The gel was then polymerized for 15 min, after which it was transferred into a centrifuge. After centrifugation, the water was removed and the cavities formed were coated in succession with ECM and colonized with the hair follicle cells.