Culture system and media for skin explants providing enhanced viability and enabling molecular studies
Inventors
OSKERITZIAN, CAROLE A. • CHUMANEVICH, ALENA P.
Assignees
Publication Number
US-12258586-B2
Publication Date
2025-03-25
Expiration Date
2039-10-21
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Abstract
Embodiments of the invention include systems and media for mammalian cell culture that enhance cell viability which enables biological assays and quantitative studies in tissues such as skin explants. Certain embodiments may be used in coordination with each other or may be practiced separately. An exemplary embodiment of the invention is a culture medium that includes Dulbecco's Modified Eagle's Medium, X-VIVO medium, and subcutaneous adipocyte medium. In one embodiment the culture medium includes approximately 30-60% Dulbecco's Modified Eagle's Medium by volume, approximately 30-60% X-VIVO medium by volume, and approximately 2.5-15% subcutaneous adipocyte medium by volume. Certain embodiments of the culture medium can also include recombinant human stem cell factor. Embodiments of the invention disclosed here are not limited to the brands described and encompass equivalents, as well as combinations, of the individual chemical components that make up the media described herein.
Core Innovation
The invention provides systems and media for mammalian cell culture that enhance cell viability in tissues such as skin explants, enabling their use in biological assays and quantitative studies. A key embodiment is a culture medium containing specific volumetric ratios of Dulbecco's Modified Eagle's Medium, X-VIVO serum free medium, and subcutaneous adipocyte medium, with possible inclusion of recombinant human stem cell factor. The culture medium is designed with approximate volume ranges of 30-60% Dulbecco's Modified Eagle's Medium, 30-60% X-VIVO medium, and 2.5-15% subcutaneous adipocyte medium, optionally supplemented by recombinant human stem cell factor.
The system can include a vessel configured to maintain defined temperature and CO2 concentration, as well as an explant cover, which may contain an antigen such as ovalbumin to stimulate an immune response. The culture medium partially immerses the skin explant, typically covering all layers except the epidermis, and the vessel maintains a controlled environment for optimal explant viability. The explant cover may serve both as a barrier and as a means to deliver chemicals or antigens to the exposed skin surface.
The problem addressed is that existing cell culture media are inadequate for maintaining skin explant viability for extended periods, thus restricting cellular and molecular studies in human skin, especially those involving mast cells. Current solutions fail to support explant viability past seven days, often necessitating reliance on animal models, which increases cost and delays human-relevant study outcomes. This invention enables extended in vitro maintenance of skin explants—up to or exceeding seven days—facilitating molecular and cellular analyses directly on human tissue.
Claims Coverage
There are two independent claims in this patent, each introducing distinct inventive features regarding the culture system and method for in vitro study.
Mammalian skin explant culture system with defined media and explant cover containing antigen
A culture system comprising: - A culture medium including Dulbecco's Modified Eagle's Medium, a serum free medium, subcutaneous adipocyte medium, and recombinant human stem cell factor at a concentration from about 150 ng/mL to about 250 ng/mL. - A vessel for culturing the explant. - An explant cover comprising an antigen present in the cover from about 1 mg/mL to about 10 mg/mL. This system supports the culture of mammalian skin explants with provision for both tailored media and antigen delivery.
Method for in vitro study on skin explant after extended viability using the specific culture system
A method of performing an in vitro study on a mammalian skin explant, wherein the study is performed after the explant remains viable in culture for at least three days using the described culture system comprising a human skin explant. The method encompasses carrying out the in vitro study following this extended viability period and leverages the components and environment specified in the culture system.
The claims cover a specifically composed culture system for maintaining and stimulating mammalian skin explants and a method for conducting in vitro studies on explants benefiting from this system's capacity to sustain viability for at least three days.
Stated Advantages
Enhances cell viability in mammalian skin explants, enabling maintenance for at least 3, 7, or more days in culture.
Facilitates cellular and molecular studies, including gene and molecular expression assays, directly on human tissue.
Reduces cost and time by potentially circumventing the need for animal models in preliminary mast cell studies.
Allows for identification and assessment of mast cell activation status and immune responses in explant cultures.
Documented Applications
In vitro studies on human skin explants, including mRNA analysis and protein analysis after extended culture.
Identification and study of mast cells and their activation status in skin explants upon exposure to chemicals or antigens.
Gene and molecular expression analysis following exposure of the epidermis to various treatments.
Assessment of skin structure preservation and changes, such as measuring epidermal thickness after culture with antigens.
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