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Sebocyte cell culturing and methods of use

A sebocyte, cell-based technology for use in cell culture active agents, cell culture supports/coatings, botanical equipment and methods to address issues such as limiting the use of cell cycle and differentiation regulation

Inactive Publication Date: 2016-01-13
CHILDRENS HOSPITAL MEDICAL CENT CINCINNATI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Human sebocyte cell lines have been established in the past from adult facial skin and the periauricular region [11-14], but their use of simian virus-40 large T antigen or HPV16 / E6E7 genes (which bypass p53 and retinoblasts) Oncoprotein-mediated checkpoint) immortalization of cells leads to transformation, which limits their use for analysis of their cell cycle and differentiation regulation

Method used

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  • Sebocyte cell culturing and methods of use
  • Sebocyte cell culturing and methods of use
  • Sebocyte cell culturing and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] [Example 1: Method for cultivating sebocytes]

[0049] Sebaceous gland clusters were generated from the scalp (SSG3), face and breasts of male and female donors aged 9 months to 12 years. Skin samples collected as surgical waste provided information on the age and sex of the donors with approval from the Institutional Board (IRB) of the Cincinnati Children's Hospital Medical Center.

[0050] After skin samples were cut into small pieces, samples were treated with 1X dispase (2 mg / ml in 1X PBS, Gibco / InvitrogenCat #17105-04; Carlsbad, California) overnight at 4°C prior to dissection. Dispase was used to separate the epidermis from the dermis and to avoid intermingling of epidermal cells.

[0051] After treating the skin with 1X dispase ( figure 1 ), using microsurgical instruments, the intact sebaceous glands were isolated under a dissecting microscope. The hair shaft and a small amount of tissue with sebaceous glands are preserved to preserve the microenvironment aro...

Embodiment 2

[0056] [Example 2: Characterization of cultured sebocytes]

[0057] 【method】

[0058] 【Western Blot】

[0059]Proteins were separated by electrophoresis on 10-12% acrylamide gels, transferred to nitrocellulose membranes and subjected to immunoblotting. Membranes were blocked for 1 h with 5% skim milk or 5% BSA in PBS containing 0.1% Tween-20. Typically the primary antibody is used at a concentration of 1 / 1,000 and the HRP-conjugated secondary antibody is used at 1 / 2,000 in 5% skim milk. Immunoblots were developed by using standard ECL (Amersham, Pittsburgh, PA) and Luminata™ crescendo and classico (Millipore). 2-color immunoblot detection was performed by using LI-COROdysseyCLx (LI-CORBiosciences, Lincoln, Nebraska). Membranes were blocked in Odyssey blocking buffer (LI-COR) and using secondary antibodies conjugated to IRDye680LT and 800CW (1 / 10,000; LI-COR). Protein levels were quantified by using an Odyssey infrared imaging system (LI-COR).

[0060] 【Retrovirus infectio...

Embodiment 3

[0113] [Example 3: Screening of Compounds]

[0114] Primary sebocytes are used to test compounds known to be inhibitors or activators of adipogenesis, and to identify compounds that test to inhibit or activate adipogenesis, or alter or alter the effect of an inhibitor or activator of adipogenesis.

[0115] Known inhibitors or activators of lipogenesis:

[0116] Androgens: sebum production under androgen control, and abnormal pilosebaceous unit response to androgens appear to be implicated in the pathogenesis of acne

[0117] 5α-reductase inhibitor (used in androgenetic alopecia): reduces lipogenesis

[0118] 5α-DHT (di-hydrotestosterone) (sebaceous gland-stimulating activity of androgens in vivo): increases proliferation, increases lipogenesis.

[0119] DHEA (5-dehydroepiandrosterone) (which is the main steroidal product secreted by the adrenal gland, acts on androgen receptors, androgenic effect on sebaceous gland activity): increases lipogenesis

[0120] Cyproterone aceta...

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Abstract

Methods of culturing sebocyte cells, isolated populations of sebocytes, and methods of using the cultured sebocyte cells for screening compounds that inhibit or activate lipogenesis are provided.

Description

【Technical field】 [0001] The present invention relates to methods for culturing sebocytes, and methods for screening compounds that inhibit or activate lipogenesis using the cultured sebocytes. 【Background technique】 [0002] In humans, sebaceous glands are distributed throughout the skin, and are found in greatest abundance on the face and scalp, and are absent only on the palms and soles. Sebaceous glands are microscopic glands that secrete an oily substance (sebum) in hair follicles to lubricate the skin and hair of animals [1]. They function with the epidermis to prevent dehydration of the skin and protect the body against infections and physical, chemical and thermal attacks of the environment. The main components of human sebum are triglycerides and fatty acids (57.5%), wax esters (26%) and squalene (12%) [2]. Sebum production is regulated throughout life and decreases dramatically with age [3]. This is associated with increased dryness and brittleness of the skin. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A01N63/00
CPCC12N5/0625C12N2501/15C12N2533/52C12N5/0633C12N2500/32C12N2500/84C12N2501/11C12N2501/30C12N2501/33C12N2533/12C12Q1/6883C12Q2600/136C12Q2600/158G01N33/5023G01N33/92
Inventor G·高舍A·J·麦克奈尔恩
Owner CHILDRENS HOSPITAL MEDICAL CENT CINCINNATI
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