# US 10,865,380 B2 · Method for improving the culture conditions of cultures of primary sweat gland cells and/or three-dimensional sweat gland equivalents

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- Number: US 10,865,380 B2
- Document ID: US10865380B2
- Status: Granted
- Kind: B2
- Application number: 15/834,069
- Filed: 2017-12-07
- Published: 2018-06-14
- Granted: 2020-12-15
- Earliest claimed: 2016-12-12
- Assignees: Henkel AG & Co. KGaA
- Applicants: Henkel AG & Co. KGaA
- Inventors: Patricia Klaka; Bernhard Banowski; Sabine Gruedl; Thomas Welss
- Web page: https://patentgrep.com/patent/US10865380B2

## CPC

- C12N 5/0633: {Cells of secretory glands, e.g. parotid gland, salivary glands, sweat glands, lacrymal glands}
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/80: Neurotransmitters; Neurohormones
- C12N 2513/00: 3D culture
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure relates to a method for improving the culture conditions of cultures of primary sweat gland cells and/or three-dimensional sweat gland equivalents, where the culture with from about 500 to about 500,000 sweat gland cells and a diameter of from about 100 to about 6,000 nm is initially prepared and then cultivated in a culture medium with neurotransmitters and/or from about 11 to about 100 ng/ml of at least one growth factor. Cultivation of these cells and models in a culture medium containing at least one neurotransmitter and/or from about 11 to about 100 ng/ml of at least one growth factor leads to an increase of sweat-gland-specific marker proteins. As a result, a loss of function, which occurs with an absence of or only low expression of these markers, can be avoided.

## Claims

### Claim 1

A method of producing cultures of primary sweat gland cells and/or three-dimensional sweat gland equivalents, the method comprising:
a) preparing a culture of primary sweat gland cells and/or a three-dimensional sweat gland equivalent in a culture medium, wherein the culture of primary sweat gland cells and/or equivalent comprises from about 500 to about 500,000 sweat gland cells and a diameter of from about 100 to about 6000 micrometers (μm);
b) cultivating the culture of primary sweat gland cells and/or the three-dimensional sweat gland equivalent in the culture medium, wherein during cultivation the culture medium comprises a neurotransmitter.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the culture prepared in method step a) is the three-dimensional sweat gland equivalent culture.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the three-dimensional sweat gland equivalent prepared in method step a) comprises 1% or less by weight of a matrix compound other than matrix compounds produced by the three-dimensional sweat gland equivalent, based on a total weight of the three-dimensional sweat gland equivalent.

### Claim 4 (depends on 2)

The method according to claim 2, wherein the three-dimensional sweat gland equivalent prepared in method step a) comprises a cell type selected from the group of coil cells, clear cells, dark cells, myoepithelium cells, duct cells and mixtures thereof.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the neurotransmitter is selected from the group of αβ-adrenergic transmitters, purinergic transmitters and mixtures thereof.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the culture medium used in method step b) comprises the neurotransmitter adenosine triphosphate (ATP) as a purinergic neurotransmitter.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the neurotransmitter comprises exactly one neurotransmitter.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the neurotransmitter is a compound that bonds on an ionotropic receptor.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the culture medium used in method step b) comprises the neurotransmitter acetylcholine as a muscarinic neurotransmitter.

### Claim 10

A method of growing a culture of a three-dimensional sweat gland equivalent, the method comprising the steps of:
a) preparing the three-dimensional sweat gland equivalent in a culture medium, wherein the three-dimensional sweat gland equivalent comprises 1% or less by weight of a matrix compound other than matrix compounds produced by the three-dimensional sweat gland equivalent, based on a total weight of the three-dimensional sweat gland equivalent; and
b) cultivating the three-dimensional sweat gland equivalent in the culture medium, wherein during cultivation the culture medium comprises a neurotransmitter and/or a growth factor.

### Claim 11 (depends on 1)

The method of claim 1, wherein:
cultivating the cell culture of primary sweat gland cells and/or the three-dimensional sweat gland equivalent in the culture medium comprises cultivating the cell culture wherein the culture medium comprises exactly one neurotransmitter during the cultivation.

### Claim 12 (depends on 9)

The method of claim 9 wherein:
the neurotransmitter comprises exactly one neurotransmitter.

### Claim 13 (depends on 1)

The method of claim 1 wherein:
cultivating the cell culture under step b) comprises cultivating the cell culture wherein the culture medium further comprises a growth factor during the cultivation.

### Claim 14 (depends on 1)

The method of claim 1 wherein:
cultivating the cell culture of primary sweat gland cells and/or the three-dimensional sweat gland equivalent in the culture medium comprises cultivating the cell culture and/or the three-dimensional sweat gland equivalent wherein the culture medium during cultivation comprises the neurotransmitter in a total concentration of from about 2.0 micromolar (μM) to about 25 millimolar (mM).

### Claim 15 (depends on 13)

The method of claim 13 wherein:
the culture medium comprises the growth factor in a total concentration of from about 11 nanograms per milliliter (ng/ml) to about 40 ng/ml during the cultivation.

### Claim 16 (depends on 13)

The method of claim 13 wherein;
the culture medium comprises the growth factor in a total concentration of from about 15 nanograms per milliliter (ng/ml) to about 25 ng/ml during the cultivation.

### Claim 17 (depends on 10)

The method of claim 10 wherein:
cultivating the three-dimensional sweat gland equivalent comprises cultivating the three-dimensional sweat gland equivalent wherein, during cultivation, the culture medium comprises a neurotransmitter.

### Claim 18 (depends on 17)

The method of claim 17 wherein:
the neurotransmitter comprises adenosine triphosphate (ATP) as a purinergic neurotransmitter, and wherein the neurotransmitter comprises exactly one neurotransmitter.

### Claim 19 (depends on 17)

The method of claim 17, wherein:
the neurotransmitter is selected from the group of αβ-adrenergic transmitters, purinergic transmitters and mixtures thereof.

### Claim 20 (depends on 10)

The method of claim 10 wherein:
the three-dimensional sweat gland equivalent is a cell model of glandular cells which has an extent in all three spatial directions.