WO2021121805A1 - Reconstruction of an eccrine sweat gland, which is supported by tissue engineering - Google Patents

Abstract

The present invention relates to a method for producing a three-dimensional model of the human eccrine sweat glands, to the models obtained in this manner, to the use of the models for product tests of antiperspirants or deodorants and to the use of the models as a transplant.

Description

Reconstruction of an eccrine sweat gland supported by tissue technology

The present invention relates to a method for producing a three-dimensional model of the human eccrine sweat glands, the models obtained in this way, the use of the models for product testing of antiperspirants or deodorants and the use of the models as a transplant.

Washing, cleaning and caring for one's own body represent a basic human need. The ongoing elimination or reduction of body odor and armpit wetness is particularly important. Armpit wetness and body odor are caused by secretion from eccrine and apocrine sweat glands in the human armpit. While the eccrine glands serve to thermoregulate the body and are responsible for the development of armpit wetness, the apocrine glands secrete a viscous secretion in response to stress, from which unpleasant body odor develops through bacterial decomposition.

The eccrine sweat gland, especially the human eccrine sweat gland, is one of the unbranched, twisted tubular glands and can be found in the secretory end piece (also called coil), the dermal duct (also called duct) and the epidermal duct (also called acrosyringium) subdivide. The cells present in these gland sections have different tasks and functions, such as secretion in the coil, reabsorption of ions in the duct and release of the secretion, in particular sweat, to the surrounding skin through the acrosyringium. With a view to avoiding underarm wetness and / or body odor, it is desirable to reduce and / or avoid secretion from eccrine and / or apocrine sweat glands.

This can be achieved, for example, by blocking the ducts of the eccrine sweat glands by means of so-called plugs.

For this purpose, in the prior art, antiperspirant aluminum and / or aluminum-zirconium salts are used, which, however, are perceived as critical by the consumer. Furthermore, antibacterial agents are used in the prior art, which prevent the bacterial breakdown of sweat. However, such agents can negatively affect the natural microflora of the skin under the armpit. It is therefore desirable to provide cosmetic agents which are capable of reliably preventing underarm wetness and / or body odor and which are free from aluminum and / or aluminum-zirconium salts and compounds with an antibacterial effect.

One possibility for providing such agents is to use substances which effectively inhibit the stimulation and / or the biological processes of the sweat glands and thus reduce or avoid sweat secretion. In order to be able to identify such substances, in-vivo tests can be carried out with test participants. Such tests are however complex and do not allow screening with high throughput rates. Alternatively, in vitro tests using cell models of sweat glands can be used, on which the influence of test substances on the stimulation of the sweat glands can be examined. In order for the test results obtained in-vitro to be transferable to the in-vivo situation, the cell model of the sweat gland used must reproduce the in-vivo situation as precisely as possible. For this purpose, three-dimensional cell models are required, since two-dimensional models do not have sufficient physiological proximity to the native sweat gland and thus only inadequately reflect the in-vivo situation. In addition, an explanation of the sweat secretion mechanism is necessary. Only in this way can so-called biological targets, in particular proteins produced by the sweat gland cells, be identified, the influence of which by test substances leads to reduced sweat production.

So far, sweat glands from isolated cells have only been cultivated as organoid structures through direct incorporation into Matrigel or as spheroids. For example, Li et al. (Three-dimensional culture and Identification of human eccrine sweat glands in Matrigel basement membrane matrix; Cell Tissue Res (2013) 354: 897-902) a reconstructed sweat gland model that includes isolated sweat gland cells in Matrigel. Matrigel forms a kind of extracellular matrix. However, such a model cannot represent the natural environment of the sweat glands, since Matrigel has a different composition than the skin. Thus, the sweat gland cells are not in a physiological tissue environment. However, in order to transfer in-vitro results to the in-vivo situation, the in-vitro model should be designed as similar as possible, ideally the same, to the in-vivo conditions.

The eccrine sweat glands are relevant for the thermal balance of the human body. The body temperature is controlled by the production of sweat. The sweat also ensures the suppleness of the skin and its correct pH value, since sweat is slightly acidic. The sweat also secretes substances such as dermcidin with an antibacterial protective effect. In the case of severe injuries to the skin, such as burns, scalds or chemical burns, there are no sweat glands in the area of the scar tissue, which means that the functions mentioned are no longer ensured in these areas. It would be desirable if sweat glands could be re-implanted in these areas. However, this has not yet been possible.

There is therefore a need for a model in which sweat glands can be reconstructed in conditions that are as similar as possible to the in vivo conditions, in order, on the one hand, to investigate active substances for use in cosmetic and pharmaceutical products. It should be possible to standardize the model and produce it inexpensively. Investigations with this model with regard to possible active substances should be able to be carried out quickly and the results should be transferable to the in vivo situation. On the other hand, there is also a need for skin grafts with contained sweat glands for use in the care of (large-scale) Injuries to the skin involving the sweat glands, such as burns, scalds or chemical burns.

It has surprisingly been found that with a model in which sweat glands are introduced into a 3-dimensional collagen matrix which contains fibroblasts, it is possible to reconstruct sweat glands in a dermis-like structure. This allows the sweat gland cells to be placed in a quasi-physiological environment with collagen and fibroblasts. Due to the dermis-like structure, a model according to the invention can be used to investigate active ingredients for use in cosmetic and pharmaceutical products. These results can be transferred well to the in-vivo situation. On the other hand, the reconstructed, skin-like models can be seen as an important step in the development of medical transplants with contained sweat glands for use in the case of burns, scalds, chemical burns or comparable large-scale injuries to the skin.

In a first embodiment, the object on which the present invention is based is therefore achieved by a method for producing a three-dimensional model of the human eccrine sweat glands comprising the following steps: a) providing a collagen-containing suspension, b) adding fibroblasts to this suspension, and then e) introducing a stamp into the suspension which contains collagen and fibroblasts, the stamp having a base surface and one or more pegs, f) hardening the suspension and g) removing the stamp, thereby creating a three-dimensional matrix of collagen and fibroblasts obtained, wherein the matrix has depressions, h) introduction of cells of the secretory part of a sweat gland, and i) introduction of cells of the reabsorptive duct of a sweat gland and k) culturing the cells.

In a further embodiment, the object on which the present invention is based is achieved by a model of the human eccrine sweat glands obtainable by the method described above. In a further embodiment, the object on which the present invention is based is achieved by using a model as described above for product testing of antiperspirants or deodorants, in particular with regard to effectiveness, undesirable side effects, irritation, toxicity and inflammation effects or allergy-triggering effects or compatibility of substances. The embodiments are further described below. Here, advantageous configurations relate to all embodiments. The features described can be combined with one another in any way. A sweat gland, which is to be simulated with the method according to the invention, comprises a secretory part, which is also referred to as a coil, and a reabsorptive duct. In connection with the present invention, the term "culturing" means maintaining the vital functions of cells, for example fibroblasts or keratinocytes, preferably in vitro, in a suitable environment, for example with the supply and removal of metabolic products and products, in particular also an increase in cells. In connection with the present invention, fibroblasts are understood to mean naturally occurring fibroblasts, in particular fibroblasts occurring in the dermis, genetically modified fibroblasts or fibroblasts resulting from spontaneous mutations or their precursors.

The fibroblasts can be of animal or human origin.

The collagen is particularly poorly soluble collagen. According to the invention, “poorly soluble collagen” is understood to mean coarse-fiber collagen which shows no visible or only slight swelling or no or only slight gel formation, in particular no gel formation, in the aqueous medium over several hours. Poorly soluble collagen is preferably obtained from the tendons of animals, in particular mammals, preferably rats, horses, pigs or cattle, very particularly preferably from the tails of rats. The collagen suspension from step a) can advantageously be poured and pipetted at room temperature without any problems. The fibroblasts are added to the collagen suspension. The collagen suspension containing fibroblasts is then placed in hardening containers, the dimensions of which correspond to those of the desired matrices. Suitable containers are, for example, Petri dishes or 6-well or 12-well plates.

The punch is inserted vertically into this suspension. The stamp has a base and pegs. The base of the stamp can be designed as desired. Its size should roughly correspond to the size of the container in which the collagen-fibroblast suspension is located. In use, the base has a top and a bottom. On the upper side, the stamp can have a device with which it can be held and guided. The stamp has at least one pin on the underside. The stamp is now inserted vertically into the suspension in such a way that the pin (s) are completely immersed in the suspension of collagen and fibroblasts. The pegs should not touch the bottom of the container. Rather, it is necessary that there is still suspension between the pin and the container, which then also hardens to form a matrix. The base of the stamp can touch the surface of the collagen-fibroblast suspension. The suspension can then harden.

The curing takes place preferably at room temperature or at a temperature higher than room temperature. The temperature at which the suspension hardens is preferably from 15 ° C. to 50 ° C., preferably from 25 ° C. to 45 ° C., in particular from 30 ° C. to 40 ° C., particularly preferably from 35 ° C. to 40 ° C. Temperatures could damage the fibroblasts. At If the temperature is too low, the curing time is too long for economic reasons. A suitable curing time is about 30-200 minutes, in particular 60100 minutes. The collagen-containing suspension provided in step a) forms a newly constructed collagen structure. The concentration of collagen is about 150 mg of collagen per milliliter of suspension, preferably 2-40 mg, in particular 3-20 mg, preferably 4-10 mg of collagen per milliliter of suspension. This corresponds to a proportion of around 0.1% to 8% collagen. The range from 0.5% to 5%, preferably from 0.8% to 2.5%, in particular from 0.8% to 1.2%, is preferred.

The fibroblasts are added to the collagen suspension (collagen gel). In a preferred embodiment, the number of fibroblasts per milliliter of collagen gel is 100 to 500,000, in particular 1,000 to 400,000, preferably 10,000 to 300,000, preferably 50,000 to 250,000, particularly preferably 100,000 to 200,000.

Since the collagen suspension is in the form of a gel, the terms collagen suspension and collagen gel are used synonymously in the following. The amount of collagen and fibroblasts is selected so that a matrix can be obtained which has skin-like properties and, in addition, curing is possible under the aforementioned conditions. The fibroblasts are preferably cultivated in a solution containing a cell culture medium (preferably DMEM cell culture medium) and serum (preferably fetal calf serum (FBS), normal calf serum (NCS), normal lamb serum (NLS), defined serum or serum substitute products). If necessary, further constituents, such as antibiotics, can be added. The stamp, which is now introduced into the suspension of collagen gel and fibroblasts, has, as already described, a base and one or more pegs. The pegs protrude from the base. The pegs penetrate the suspension and after the suspension has hardened, cavities are formed in the matrix. In terms of their spatial configuration, these cavities essentially correspond to the shape of the pegs. The number of pins on the base is limited by the manufacturing process of the stamp and the size of the base and the size of the pins.

Since it is an object of the invention to provide such matrices as possible that simulate the skin surface, the number and size of the pegs (length and diameter) is selected in such a way that it is modeled on the skin. The number of sweat glands in the skin fluctuates significantly in the human body, so that, depending on the planned application, different densities of cones can be selected on the base of the stamp. The diameter of the pins is preferably in the range from 100 μm to 2 mm, in particular from 200 μm to 1.5 mm, preferably from 500 μm to 1 mm. The number of pins on the base of the stamp is in particular in the range from 1-300, preferably from 5-200, in particular from 10-100. The length of the pins is preferably in the range from 1 mm to 10 mm, in particular from 2 mm to 8 mm, in particular from 3 mm to 6 mm. This length is suitable for replicating sweat glands as naturally as possible. The shape of the pin is preferably such that the diameter is constant over the entire length of the pin. Particularly preferably the diameter is over 95% of the Length, preferably constant over 90% of the length, in particular over 85% of the length or 80% of the length. If you look at the pin on the base, the area that adjoins the base is constant. The free end of the pin can have a smaller or a larger diameter. For example, the pin can taper constantly towards the end so that a kind of tip is formed. It is also possible for the free end of the pin to be designed in the manner of a small ball, so that a type of trough later results in the matrix. The free end of the pin preferably has a smaller diameter than the remaining length of the pin, since otherwise the structure obtained can be damaged when the stamp is removed from the matrix.

In order to obtain a stamp with the highest possible density of pins with a constant design, the stamp can be produced, for example, by means of 3D printing. The stamp is preferably made from a plastic. It must be ensured that the plastic has a surface that is as smooth as possible so that it can be removed from the matrix after hardening without damaging the matrix. For example, "Durable Resin", which is manufactured by formlabs GmbH, Berlin, is suitable. After the stamp has hardened in the matrix and removed from it, the matrix represents the negative of the stamp, so to speak. This results in a matrix of collagen and fibroblasts which has cavities. The number and dimensions of the cavities correspond to the pins on the stamp. According to the invention, cells of the secretory part of a sweat gland, that is to say cells of the coil, are first introduced into these cavities. The number of cells of the secretory part per cavity is preferably in the range from 100-500,000, in particular in the range from 250-350,000, preferably from 500-200,000, particularly preferably from 1000-100,000. The number of cells depends on the configuration of the pins and thus on the volume of the cavity. The number of cells of the secretory part should be chosen so that a cavity is formed in the middle to mimic the function of the sweat glands. The cells of the secretory part of the sweat gland are cultivated in the cavities of the matrix. For this purpose, the cells of the secretory part are preferably taken up in K medium and introduced into the cavity, for example with a pipette. Then cells of the reabsorptive duct are introduced into the cavities of the matrix. For this purpose, the duct cells are preferably taken up in K medium and introduced into the cavity, for example with a pipette. Before the cells of the reabsorptive duct are introduced into the cavities of the matrix, the cells of the secretory part of the sweat glands are cultivated for a sufficiently long period of time. This period of time is preferably 3-24 hours, in particular 6-18 hours, preferably 8-16 hours. For example, the cultivation can take place overnight. A sufficiently long cultivation is necessary to ensure the formation of a cell network that resembles the coil. Then cells of the reabsorptive duct are added. This is followed by another cultivation in order to enable the formation of a sweat gland structure. A layer of 1 to 5 is formed Rows of cells (monolayer or multilayer) at the edge of the cavities in the matrix, but without completely filling the cavity.

The cultivation time is preferably three days to 26 weeks, preferably seven days to twelve weeks, in particular ten days to eight weeks, particularly preferably 14 days to four weeks. The cultivation takes place over a period of time that is necessary for the cells to differentiate and the desired sweat gland structure to develop. The cells are preferably cultivated in a specific nutrient medium. A particularly good cultivation of the duct cells and coil cells is achieved if K medium is used. This contains medium components (DMEM GlutaMax I and HAM F12), serum components (10% Fetal Clone II), nutrients (4 mM L-glutamine, 0.12 UI / ml insulin, 2.43 pg / ml adenine, 1 mM L-ascorbine - acid phosphate), growth factors (10 ng / ml Epithelial Growth Factor (EGF), 0.4 pg / ml hydrocortisone, 10-10 M cholera toxin, 2 * 10-9 M (5 pg / ml) 3.3 ', 5- Triiodo-L-thyronine) and antibiotics (100 ul / ml penicillin G, 25 pg / ml gentamicin solution). The cultivation is preferably carried out in this medium at a temperature of 36 ° C. to 38 ° C. and a CO 2 content of 5% by weight, based on the total weight of the atmosphere used for cultivation. After a sufficient incubation time, three-dimensional structures are formed in the cavities of the matrix, which are modeled on those of the human sweat glands. In addition to collagen, the matrix has fibroblasts. The fibroblasts reduce the stability of the matrix, but the presence of fibroblasts is necessary to ensure cell-cell communication that corresponds to the natural conditions. The method according to the invention thus offers the possibility for the first time to provide a model which is a connection of a dermis-near fibroblast-collagen matrix with sweat gland cells in order to simulate the physiological situation in the skin. In addition to the cell-cell contacts, the shape of the sweat glands can be specified in the models and the situation in the human skin can be simulated through the preferred use of stamps printed by means of 3D printers.

The present invention is explained below by way of example without, however, being restricted thereto.

Example:

A collagen mixture was prepared on ice in a petri dish. For this purpose, 700 μl 10x PBS (Phosphate Buffered Saline) were presented. To adjust the pH, 78 μl of 1 M NaOH and 3400 ml of collagen type I solution (amount of collagen in collagen mixture: 4 mg / ml) were added. The total amount was 4178 ml. 125,000 fibroblasts per milliliter of collagen suspension were added to this collagen suspension, which is in the form of a type of gel. Subsequently, 700 ml of the collagen-fibroblast suspension were placed in each insert of a 12-well plate. A stamp was produced from "Durable Resin" by the company formlabs GmbH, Berlin, Germany, by means of 3D printing. The punch had 16 pegs, with four pegs being arranged in a row. The diameter of the pegs was 0.8 mm and the length 4 mm. The selected length ensured that when the cones were fully immersed in the collagen-fibroblast suspension, the distance between the cones and the bottom of the plate was 2 mm.

The stamp was immersed in the suspension. After about 1.5 hours at 37 ° C., the stamp could be removed. A matrix with 16 cavities was obtained. Coil cells were sown with a density of 1.2 * 10 6 cells, trypsinized and taken up in K medium. Approximately 5,200 cells were pipetted into each well of the matrix. The incubation took place in K-medium overnight at 37 ° C. Duct cells were sown with a density of 1 * 10 6 cells, trypsinized and taken up in K-medium. Approximately 54,000 duct cells were pipetted into each well of the matrix. Incubation took place at 37 ° C. Further cultivation was also carried out at 37 ° C., with a change of medium in each case after 2 or 3 days. After 4 weeks a sweat gland equivalent had developed. All cultivations / incubations took place in a 5% CO 2 atmosphere.

Claims

Claims 1 . A method for producing a three-dimensional model of the human eccrine sweat glands comprising the following steps: a) providing a collagen-containing suspension, b) adding fibroblasts to this suspension, and then e) inserting a stamp vertically into the collagen-fibroblast susceptible thus obtained - pension, wherein the stamp has a base and one or more pegs, such that the pegs are immersed in the suspension, f) hardening of the suspension and g) removal of the punch, whereby a three-dimensional matrix of collagen and fibroblasts is obtained, the Matrix has wells, h) introduction of cells of the secretory part of a sweat gland, and i) introduction of cells of the reabsorptive duct of a sweat gland and k) culturing the cells. 2. The method according to claim 1, characterized in that the concentration of collagen in the collagen-containing suspension is 1 mg / ml to 50 mg / ml, in particular 2 mg to 40 mg per milliliter of collagen-containing suspension. 3. The method according to claim 1 or 2, characterized in that the number of fibroblasts is 100 to 500,000, preferably 1000 to 400,000, in particular 10,000 to 300,000 per milliliter of collagen-fibroblast suspension. 4. The method according to any one of claims 1 to 3, characterized in that the punch has 1 to 300, preferably 5 to 200, in particular 10 to 100 pins. 5. The method according to any one of claims 1 to 4, characterized in that the diameter of the pin is in the range from 100 μm to 2 mm, in particular from 200 μm to 1.5 mm, preferably from 500 μm to 1 mm. 6. The method according to any one of claims 1 to 5, characterized in that the length of the pin is from 1 mm to 10 mm, in particular from 2 mm to 8 mm, preferably from 3 mm to 6 mm. 7. The method according to any one of claims 1 to 6, characterized in that the stamp is produced by means of 3D printing. 8. The method according to any one of claims 1 to 7, characterized in that the number of cells of the secretory part of a sweat gland which are introduced in step h) is 100 to 500,000, preferably 250 to 350,000, in particular 1,000 to 100,000. 9. The method according to any one of claims 1 to 8, characterized in that the number of cells of the reabsorptive duct of a sweat gland which are introduced in step i), 100 to 500,000, preferably 250 to 350,000, in particular 1,000 to 100,000. 10. The method according to any one of claims 1 to 9, characterized in that the cultivation in step k) is 3 days to 26 weeks, in particular 7 days to 4 weeks. 11. Method according to one of Claims 1 to 9, characterized in that the cultivation in step k) takes place in K medium. 12. Model of the human eccrine sweat glands obtainable by a method according to one of the preceding claims. 13. Use of a model according to claim 12 for product testing of antiperspirants or deodorants, in particular with regard to effectiveness, undesirable side effects, irritation, toxicity and inflammation effects or allergy-inducing effects or compatibility of components. 14. Use of a model according to claim 12 as a medical skin transplant.