Hair follicles are known to contain a well-characterized niche for adult stem cells including the bulge containing epithelial and melanocytic stem cells. Stem cells in the hair bulge can generate the interfollicular epidermis, hair follicle structures and sebaceous glands. The budge epithelial stem cells can also reconstitute in an artificial in vivo system to a new hair follicle. It has been known that transplanting hair follicle stem cells in balding scalp results in increase hair density and may promote new formation of hair follicles. Stem Cell Investig. 2017, 4:58. However, the result of the transplantation was somewhat limited as the amount of the transplanted stem cells was limited to the amount obtained from a patient through a physical separation. For increased effectiveness of such transplantation, a method and a system that allows culturing such stem cells in vitro in particularly in 3D. Also, after transplantation of the stem cells, adjusting planted scalp conditions for better settlement of the stem cells would help the effectiveness of such treatment.
3D cell culture techniques have led to the creation of more predictive in vitro cell models for numerous applications including cancer research, drug discovery, neuroscience and regenerative medicine. Cells naturally grow and differentiate in three dimensional environments. Cells in their natural environment have constant interactions with extracellular matrix proteins (ECM) and other cells, regulating complex biological functions like cellular migration, apoptosis, or receptor expression. Most of these interactions are lost, or significantly reduced, in traditional 2D cell cultures. Advanced 3D cell systems allow researchers to bridge the gap between classical 2D cell culture and in vivo animal models. Recently, the use of advanced 3D cell culture methods such as tumor spheroids, stem cell organoids and tissue engineering via 3D bioprinting have been implemented to more closely model real in vivo cellular responses. Improving 3D cell culture models to accurately replicate the natural environment will provide more meaningful scientific conclusions and ultimately improve human health. 3D cell culture models can be divided into two main categories: 1) scaffold-based methods using animal derived/synthetic hydrogels or structural 3D scaffolds and 2) scaffold-free approaches using freely floating cell aggregates termed spheroids.
Existing 3D methods are not without limitations, including scalability, reproducibility, sensitivity, and compatibility with high-throughput screening (HTS) instruments. Growing 3D cell models take long time to be utilize as short-term clinical test uses. Also, maintaining 3D cell models in vitro for a long period of time is challenging. Accordingly, a new method and system for 3D cell culture is needed. A 3D cell model which can be cultured in a short period of time can be used for a patient specific medical treatment and can improve efficiency of drug discovery.
The disclosed embodiments of the present disclosure are directed to overcoming one or more of the problems with existing 3D culture methods and systems and illustrate the present invention. The scope of the invention shall not be limited to the disclosed embodiment.
One embodiment of the present invention relates to a method for producing 3D cell mass efficiently and effectively using a secondary cell line to support the rapid growth of a hair follicle stem cell to be grown to a three-dimensional structure or three dimensional mass of cells, e.g. organoid, which is herein defined as any 3D mass of cells or 3D cell models including conventional organoid and spheroid as well as tissue. Such 3D cell models have many benefits over conventional two-dimension cell structures. For example, 3D organoid is physiologically more like actual biologic organ or tissue and can be used for drug discovery or screen or medical treatments such as cell therapies, etc. 3D organoid can be used for creating micro physiological systems or organs-on-chips to study difficult clinical problems and development of potential treatments. These human surrogates make use of pumpless self-contained systems that are robust, easy to use, and low cost and allow measurement of metabolic and functional responses.
In particular, one embodiment provides a method for culturing hair follicle stem cells using patient derived hair follicle stem cells cultured by using a feeder cell. Where the feeder cell in a medium may be provided in a way that the feeder cell medium is separated from the medium for hair follicle stem cells. A medium with various growth factor may be supplied on the top of the feeder cell medium and the hair follicle stem cell medium. The resulting culture bed is incubated
The feeder cell may be a dermal endothelial cell or a fibroblast cell, preferably a dermal endothelial cell from the patient. Scalp tissue may be separated from a patient. Using a single cell isolation method, dermal endothelial cells and hair follicle stem cells can be obtained from the scalp tissue. The dermal endothelial cells may be further cultured to expand its population such as 3d culture before using the dermal endothelial cells as the feeder cell.
Another embodiment provides a method for growing a 3D organoid of hair follicle stem cells by preparing a first medium with a follicle stem cell from a patient, preparing a second medium with a dermal endothelial cell from the patient, placing the first medium and the second medium in a grow substrate, placing a conditioned medium over the growing substrate to cover the first medium and the second medium, resulting in a culture plating, incubating the culture plating to grow the 3D organoid and harvesting cultured hair follicle stem cells.
Another embodiment provides a method for promoting hair growth in a patient by injecting hair follicle stems cells derived from the patent into the patient's skin where the hair follicle stems cells are harvested from a culture bed comprising a hair follicle stem and a dermal endothelial cell from the patient wherein the dermal endothelial cell is a feeder cell. In order to improve the hair growth, dermal endothelial cells may be injected along with the hair follicle stem cells.
The target cell may be placed on a separable substrate plate so that the substrate plate having the target cell can be transferred to a different culture bed. In that way, the cultured target cell can be exposed a new bed with a flash second cell or cell line. The target cell may be any cell from any part of human body such as an organ tissue or skin tissue but may be from a foreign cell such as cancer cell.
The culture substrate is a glass bottom dish where a portion of the dish bottom has a glass plate, which can be removed from the glass bottom dish. For example, the portion of the matrigel mixture over the medium containing the target cell may be removed conveniently by lifting the glass plate and relocated into another glass bottom dish to provide a fresh feeder cell. The materials for the glass plate and the glass bottom dish can vary as needed. For example, plastic material may be used.
The feeder cell or cell line is to promote the growth of the target cell and is not for own growth. The feeder cell or cell line is preferably an endothelial cell or fibroblast cell. More preferably, the endothelial cell is a human dermal microvascular endothelial cell. Even more preferably, the feeder cell or cell line is an endothelial cell or fibroblast cell from an organ that is similar to or the same as the organ from which the target cell is obtained.
The culture bed may have a second feeder cell or cell line 410 which is similarly prepared in a conditioned medium and matrigel. The second feeder cell or cell line mixture 400 may also be placed in a way that the mixture 400 is separated from the target cell or cell line mixture. The second feeder cell or cell line can be placed in the dish. The second feeder cell or cell line is preferably cell or cell lines that is similar to or the same cell or cell line as the target cell. Since the target cell mixture bed is separated from the feeder cell beds, cultured target cells can be harvested with any contamination from the feeder cell beds.
Optionally, the mixture 200 is placed on a separate removable plate 500, which allows removing the mixture bed from the dish and relocate to another dish to provide additional feeder cells or nutrients.
After the target cell and feeder cell mixtures are placed in the dish, a conditioned medium can be filled in the dish.
The conditioned medium can be chosen from various media. Many cell culture medium formulations are documented in the literature and a number of media are commercially available. Once the culture medium is incubated with cells, it is known to those skilled in the art as “spent” or “conditioned medium”. Conditioned medium contains many of the original components of the medium, as well as a variety of cellular metabolites and secreted proteins, including, for example, biologically active growth factors, inflammatory mediators and other extracellular proteins.
When the cultured target cells do not require to be separated from foreign cells from the feeder cells, the feeder cell or cell line mixtures 610 may be mixed with the target cell mixture 620 in a conditioned media 630 as the illustrated culture bed 600 in
The 3D culture method and system of the present invention can be used to culture tissues. For example,
An embodiment of the present invention can be used to culture cells or tissues for direct transplantation to a patient. For example,
The various stem cell colonies grown in 3 D culture can be used in stem cell treatment, drug discovery, drug sensitivity test, and other stem cell science. In particularly, the 3D culture methods and systems according to the present invention allow grow stem cell colonies or tissue in a shorter period of time than the conventional methods and systems. The speed is particularly important to utilize the method and system as patient-specific medicine because, for example, a cancer patient would require a quick drug screen or cell therapy as cancers usually grow very rapidly.
Also, the 3D culture systems and methods according to the present invention sustain the cultured cells or tissue for a long period of time. If necessary, the cultured cells o tissues can live several months, even longer. This is a very important feature for drug discovery or tests.
The following examples are illustrative purpose only. The scope of the invention should not be limited to these examples.
Dermal Endothelial Cells Isolation
3D Organoid Culture of Hair Follicle Stem Cells
Number | Date | Country | Kind |
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10-2018-0033594 | Mar 2018 | KR | national |
This application claims the benefit under 35 U.S.C. Section 371, of PCT International Application No. PCT/KR2019/003258, filed on Mar. 20, 2019, which claimed priority to Korean Patent Application 10-2018-0033594 filed on Mar. 22, 2018, the disclosures of which are hereby incorporated by reference herein in their entirety.
Filing Document | Filing Date | Country | Kind |
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PCT/KR2019/003258 | 3/20/2019 | WO | 00 |