Inventors: PARK, Jung-Keug, YOON, Hee-Hoon, SHIN, Youn-Ho, KIM, Young Jin; Seoul, South Korea
Publication date: 2006-01-06
Invention summary:
The present invention relates to a method for preparing a dermal papilla tissue having hair follicle inductive potency which comprises the steps of proliferating dermal papilla and lower dermal sheath cells isolated from the hair follicle in a primary culture medium known in the art, and culturing the proliferated cells in a secondary culture medium supplemented with high concentrations of amino acids, vitamins and a growth factor
Abstract:
The inventive method for the preparation of a dermal papilla tissue in accordance with the present invention makes it possible to form a quantity of the dermal papilla tissues having hair follicle inductive ability, and accordingly, it can be effectively used for the treatment of alopecia through cell transplantation
First Claim:
A method for the preparation of a dermal papilla tissue which comprises the steps of: 1) isolating dermal papilla and lower dermal sheath cells from the hair follicle;
2) culturing the isolated cells in a primary culture medium containing 600 to 1,900 m/£ of amino acids and 12 to 36 mg/£ of vitamins; and
3) inducing auto-aggregation of the cells obtained in step (2) by culturing them in a secondary culture medium containing 2,000 to 3,000 rng/^ of amino acids and 40 to 60 W>I2 of vitamins, wherein the medium is free of serum and supplemented with 0.1 to 10,000 ng/ro£ of a growth factor.
Background:
It is believed that alopecia (baldness) is hereditary and occurs when the hair follicles slowly begin to produce finer and shorter hairs, or stop producing hairs at all. There are many forms of hair loss, ranging from alopecia areata to androgenic alopecia, also known as male or female pattern baldness.
In the past, alopecia was typically treated by various methods of implanting synthetic or artificial hair into hair follicle root bulbs of the scalp, but such artificial hair implant methods have been almost always ineffective, often leading to patient health problem and further natural hair follicle loss. Currently, there are two methods employed to treat alopecia: drug therapy and human hair transplantation. The drag therapy may enhance the hair growth and prevent future hair loss, but it is associated with problems such as skin irritation and accelerated hair loss where the medication is stopped after a prolonged use. The transplantation of human hair, on the other hand, involves taking plugs of natural hair from hair growing areas and transplanting them to bald areas. The transplanted hair settles at the transplant area as a complete hair follicle and becomes a permanent hair that undergoes a normal growth cycle. However, the number of hair to be transplanted is very limited, and in case of transplanting about 2,000 hairs per one operation, it is not plausible to perform more than three such operations. Further, it is a tedious and expensive procedure that requires the expertise of a skilled physician and the motivation and tolerance of the patient. This treatment is usually not performed for females with androgenetic alopecia, since the loss of hair is too diffuse.
Thus, the currently used methods of treatment for alopecia have numerous limitations, and therefore, there have been reported many studies to overcome such problems through culturing hair follicle forming cells in vitro and implanting them.
Arase et al. have disclosed that when plucked follicles are cultured in vitro together with isolated dermal papilla cells, the hair follicle cells move toward the dermal papilla and form new hair bulbs (Arase S. et al., Skin Pharmacol. 7(l-2):12-5, 1994), which suggests that the formation and maintenance of the hair follicle are achieved by complex and intimate interactions between the outer root sheath (ORS) cells and dermal papilla cells, and if such interactions are reproduced, it is possible to re-constitute the hair follicle. Further, Cohen and Oliver have shown that dermal papilla cells play a key role in the growth of the hair follicle (Cohen J., J Emhryol Exp Morphol. 9:117-27, 1961 ; Oliver RF, J Embryol Exp Morphol. 15(3):331-47, 1966; and Oliver RF., J Embryol Exp Morphol. 18(1):43-51, 1967).
Reynolds et al. have reported that when dermal papilla cells are isolated and cultured in vitro, the dermal papilla cells gradually lose their innate hair follicle-inductive potency after about 3 to 4 passage numbers (Reynolds AJ et al., Development. 122 (10):3085-94s 1996), while Inamatsu et al. have found that when rat's hair follicle dermal forming cells are cultured in a waste medium of epithelial cells, the dermal papilla cells retain their hair follicle-inductive ability (Inamatsu M. et al., J Invest Dermatol. 111(5):767-75, 1998).
Jahoda and Reynolds have reported that as a result of isolating and culturing the dermal papilla cells of rat's vibrissa hair and implanting the cultured cells of 3 passage numbers or less into small ear skin wounds and the back of rats, abnormally large hair fibers displaying vibrissa hair-type characteristics emerge from the transplant sites (Jahoda CA., Development. 115(4): 1103-9, 1992; and Reynolds AJ. et al., Development. 115(2):587-93, 1992), and Gharzi et al. have disclosed that when the outer root sheath and lower dermal sheath of rat vibrissa hair are cultured in a collagen gel matrix which is similar to the real skin, inserted the real dermal papilla between these two cell layers, and transplanted it at the back of rat, the formation of the dermal papilla can be induced (Gharzi A. et al., Exp Dermatol. 12(2): 126-36, 2003). Further, Kevin et al. have demonstrated that the lower dermal sheath cells encompassing the dermal papilla as well as the dermal papilla cells have the hair follicle inductive ability (Kevin J. et al., J Invest Dermatol. 121: 1267-1275, 2003). Recently, the lower dermal sheath cells have been actively employed in the study for the formation of the hair follicle. The present inventors have developed an effective method for in vitro re-constitution of a dermal papilla tissue important for the growth and maintenance of the hair follicle, which comprises the steps of proliferating dermal papilla and lower dermal sheath cells isolated from the hair follicle having hair follicle inductive potency in a primary culture medium well-known in the art, and culturing the proliferated cells in a secondary culture medium containing high concentrations of amino acids and vitamins together with a growth factor. The method of the present invention makes it possible to form a quantity of dermal papilla tissues through cell auto-aggregation without the use of any matrix or substrate.
Summary:
Accordingly, it is an object of the present invention to provide a method for the in vitro preparation of a dermal papilla tissue for the purpose of forming a quantity of the dermal papilla tissues by cell auto-aggregation without the use of any matrix or substrate.
In accordance with one aspect of the present invention, there is provided a method for the preparation of a dermal papilla tissue which comprises the steps of:
1) isolating dermal papilla and lower dermal sheath cells from the hair follicle;
2) culturing the isolated cells in a primary culture medium up to 5 to 6 passage numbers, the primary culture medium containing 600 to 1,900 wgl2 of amino acids and 12 to 36 W.I2 of vitamins;
3) inducing auto-aggregation of the cells obtained in step (2) by culturing them in a secondary culture medium containing 2,000 to 3,000 W>lt of amino acids and 40 to 60 rog/i? of vitamins, wherein the secondary culture medium is free of serum and is supplemented with 0.1 to 10,000 ng/n^ of a growth factor; and
4) harvesting the auto-aggregated cells obtained in step (3) by centrifugation and culturing them in the culture medium of step (3). |
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