Method for the propagation of hair
Abstract
Method of reproduction of hair, which method comprises the
following steps: (1) removal of hair in the anagen phase from
one or more donor regions in such a way that the bulb characteristic
of hair in the anagen phase is still attached to the hair
removed, (2) culture of hair follicle cells from the hair
removed, under conditions such that the hair follicle cells
are able to multiply, and (3) implantation of the cultured
hair follicle cells in the receptor regions. Medium which
comprises at least one suitable serum-free keratinocyte culture
medium and extracts of at least one human mast cell line and
method for culturing hair follicle cells from hairs in the
anagen phase, wherein one or more hairs in the anagen phase
are placed in said medium.
| Inventors: |
Gho; Conradus Chosal
(Bunde, NL) |
| Assignee: |
Gho'st Holding B. V.
(Bunde, NL) |
| Appl. No.:
|
380580 |
| Filed: |
October 20, 1999 |
| PCT Filed: |
March 5, 1998 |
| PCT NO: |
PCT/NL98/00129 |
| 371 Date: |
October 20, 1999 |
| 102(e) Date: |
October 20, 1999 |
| PCT PUB.NO.:
|
WO98/47471 |
| PCT PUB.
Date: |
October 29, 1998 |
Foreign Application Priority Data
| Current U.S.
Class: |
424/93.7;
424/70.1 |
| Intern'l Class:
|
A01N 063/00; A01N
065/00; A61K 007/06 |
| Field of Search:
|
424/93.7,70.1 623/15
436/63 |
References Cited [Referenced
By]
U.S. Patent Documents
| 5556783 |
Sep., 1996 |
Lavker et al. |
|
| 5635387 |
Jun., 1997 |
Fei et al. |
|
| 6050990 |
Apr., 2000 |
Tankovich et al. |
|
| Foreign Patent Documents |
| 0236014 |
Feb., 1987 |
EP. |
|
| 236 014 |
Sep., 1987 |
EP. |
|
| 682 107 |
Nov., 1995 |
EP. |
|
| WO 92/00376 |
Jan., 1992 |
WO. |
|
| WO 92/07877 |
May., 1992 |
WO. |
|
| WO 95/01423 |
Jan., 1995 |
WO. |
|
| WO 96/32961 |
Oct., 1996 |
WO. |
|
Other References
Lenoir et al., Developmental Biology, 130: 610-620.
Outer root sheath cells of human hair follicle are able
to regenerate a fully differentiated epidermis in vitro,
1998.*
Schirren et al. The AM J of Dermatopathology, 19(4):
334-340. Fetal and adult hair follicle: An immunohistochemical
study of anticytokeratin antibodies in formalin-fixed
and paraffin-embedded tissue, Apr. 1997. |
Primary Examiner: Tate; Christopher R.
Assistant Examiner: Flood; Michele C.
Attorney, Agent or Firm: Sughrue Mion, PLLC
Claims
What is claimed is:
1. A method for the cosmetic reproduction of hair, which method
comprises:
(a) removing hair in the anagen phase from one or more donor
regions by plucking so that a bulb is still attached to the
hair removed, wherein the removing of hair in the anagen phase
is done by plucking the hair out of the donor region(s), and
selecting hairs in the anagen phase;
(b) culturing keratinocytes from the hair removed under conditions
whereby keratinocytes multiply, wherein the keratinocytes are
cultured in a culture medium supplemented with (i) at least
one human mast cell line and/or autologous cultured CD34.sup.+
cells, or (ii) one or more extracts of a human mast cell line(s)
and/or of the autologous CD34.sup.+ cells, and/or (iii) growth-stimulating
agents; and
(c) introducing the cultured keratinocytes into the pores of
receptor regions.
2. Method according to claim 1, wherein the keratinocytes are
cultured in a culture medium supplemented with one human mast
cell line, a subclone thereof and/or autologous cultured CD34.sup.+
cells, and/or growth-stimulating agents.
3. Method according to claim 2, wherein the human mast cell
line is one human mast cell line and/or an autologous cultured
CD34.sup.+ cell line.
4. Method according to one of the preceding claims, wherein
the keratinocytes are cultured at a temperature of 30-40.degree.
C. in an atmosphere which is saturated with water and contains
3-10% by volume of CO.sub.2.
5. Method according to claim 1, wherein a quantity of a suspension
of cultured keratinocytes and/or autologous cultured CD34.sup.+
cells is introduced into each pore whereby said quantity contains
sufficient keratinocytes and/or autologous cultured CD34.sup.+
cells to allow a hair follicle to develop.
6. Method according to claim 5, wherein the quantity of suspension
introduced per pore is 0.01-5 .mu.l.
7. Method according to claim 6, wherein the quantity is 0.1-0.5
.mu.l.
8. Method according to one of claims 5-7, wherein the suspension
is introduced into each pore via a metering pipette which is
provided with a hollow needle.
9. Method according to one of claims 5-7, wherein the suspension
is introduced via a repeating injection-metering apparatus.
10. Method according to claim 9, wherein a follicle-conducting
needle is used.
11. The method according to claim 8, wherein the hollow needle
is an 18 to 40 gauge needle.
Description
The present invention firstly relates to a method for the reproduction
of hair. The invention also relates to a method for culturing
hair follicle cells from anagenetic hairs in the anagen phase,
to a medium that is highly suitable for culturing hair follicle
cells, to a method for the preparation of such a medium and
to a precursor medium.
A method for the reproduction of hair is disclosed in European
Patent Application 0 236 014 in which epidermal follicle cells
of the desired hair type are removed from the scalp skin of
a patient. The epidermal follicle cells are then cultured in
a culture medium which preferably contains growth factors. In
a subsequent step, an opening is made in the epidermis of the
patient's scalp and, via said opening, the cultured epidermal
follicle cells are introduced into the dermis next to the epidermis.
The disadvantage of this method is that it comprises an invasive
method and that the cells are not placed directionally, as a
result of which many cells are necessary and the probability
of regeneration of hair is much lower. In this method, use is
also not made of autologous (cultured) CD34.sup.+ cells.
The essential growth structures of hair are the so-called hair
follicles, which are present in the skin. The hair follicle
cells or keratinocytes reproduce from these hair follicles and
during their path to the skin surface the cytoplasm of said
cells is converted by a large number of complex processes into
the tough and resilient material which is known as hair. The
growth cycle of hair can be subdivided into three phases: the
anagen phase or growth phase, the catagen phase or transitory
phase and the telogen phase or dying phase. The hair follicle
is unique because of the cyclic nature of hair formation and
hair growth. Specifically, the hair follicle is the only part
of the body to have a growth nucleus, from which new hairs can
be produced after removal of the old hair.
It is known that hair follicle cells from plucked human hair
can be cultured. It is also known that it is possible using
such cultured cells to form a differentiated epidermis or a
fully developed epidermis, both in vitro and in vivo. Cultured
hair follicle cells from mice can stimulate hair growth when
said cells are implanted into test animals. However, to date
it has not proved possible to achieve new hair growth in humans
in those locations where there is undesirably no (longer any)
hair with the aid of cultured autologous hair follicle cells.
People usually find baldness to be undesirable from the cosmetic
and aesthetic standpoint. However, baldness frequently occurs
and it is a known phenomenon that in particular men also become
balder as they got older. This form of baldness is known as
alopecia androgenetica. To date it is not precisely known why
certain parts of the scalp are susceptible to this alopecia
androgenetica and other parts are not. However, with women as
well it regularly occurs that the hair becomes thinner and even
threatens to largely disappear. For women in particular this
is highly undesirable from the cosmetic and aesthetic standpoint.
A known technique for combatting baldness is to transplant hair.
With this procedure hair from a donor region covered in hair,
which frequently is located on the back of the head, is removed,
including the skin, and cut into small pieces, which usually
then have only one to three hairs. These pieces are then implanted
in the bald region (receptor region). A major disadvantage of
this method is that it is at the cost or the donor region. Specifically,
hair is removed from this region and this hair does not return
again. The transplantation technique therefore offers limited
possibilities.
The aim of the present invention is to provide a technique with
the aid of which bald patches can be provided with hair again,
but which does not have the disadvantages of hair transplantation
which have been outlined above. A further aim or the invention
is to provide a method with which new hair growth on bald patches
can be achieved in humans with the aid of cultured hair follicle
cells. A further aim of the invention is to provide a method
which is relatively simple to carry out.
Said aims are achieved with the method according to the invention,
wherein hair is, as it were, reproduced. Specifically, according
to the invention the hair is removed from a donor region in
such a way that new hairs come back in its place whilst new
hair follicle cells are cultured from the hairs removed, from
which cells, in turn, new hair can form.
The invention therefore relates to a method for the reproduction
of hair, which method comprises the following steps:
(1) removal of hair in the anagen phase from one or more donor
regions in such a way that the bulb characteristic of hair in
the anagen phase is still attached to the hair removed.
(2) culture of hair follicle cells from the hair removed, under
circumstances such that the hair follicle cells are able to
multiply, and
(3) implantation of the cultured hair follicle cells in the
receptor regions,
wherein:
the hair in the anagen phase is removed by plucking hair out
of the donor region or the donor regions, followed by selection
of suitable hairs in the anagen phase,
the hair follicle cells are cultured in a culture medium which
is optionally supplemented with (a) at least one human mast
cell line and/or autologous (cultured) CD34.sup.+ cells, or
(b) one or more extracts of the human mast cell line(s) and/or
of the CD34.sup.+ cells and/or (c) growth-stimulating agents,
and
the cultured follicular hair cells and/or the autologous (cultured)
CD34.sup.+ cells are implanted in the pores of the receptor
region.
According to the invention, only one hair follicle cell or one
autologous (cultured) CD34.sup.+ cell or a combination of these
two cells can be introduced although a plurality of cells is
usually mentioned in the description.
The method according to the invention has the major advantage
that hair growth can be achieved again on bald patches without
this being at the expense of the donor region.
Step (1) of the method according to the invention comprises
the removal of hair in the anagen phase from a donor region
where such hairs are located. As explained above, hair growth
comprises three phases: an anagenous, a catagenous and a telogenous
phase. Only hairs which are in the anagenous phase are suitable
for the method according to the invention. Compared with hairs
in the catagen and telogen phases, such hairs in the anagen
phase are characterized in that they have a--frequently pigmented--bulb
of a shape characteristic for hairs in the anagen phase at the
bottom of the hair. This is generally known and to an experienced
eye a hair in the anagen phase is therefore also immediately
recognizable from the shape of the bulb. In the case of doubt
the use of a microscope can provide a definite answer. The removal
of hairs in the anagen phase can therefore be effected in various
ways, as long as the bulb characteristic of hairs in the anagen
phase is still attached to the hair removed.
The number of hairs in the anagen phase needed to culture a
suitable quantity of hair follicle cells is hardly subject to
specific limits since in principle an unlimited number of hair
follicle cells can be cultured from a single hair. Specifically,
subcultures can, in turn, be cultured from the first culture
and, in turn, several cultures can be cultured from every subculture.
In practice, 3 to 10 hairs in the anagen phase will be ample.
The hairs which ultitmately are formed in the receptor region
from the cultured hair follicle cells assume the characteristics
of the hairs from the donor region from which the hair follicle
cells were cultured. Therefore, a region which is not susceptible
to alopecia androgenetica is taken as a suitable donor region.
However, this is not essential.
Since the difference between hairs in the various growth stages
can be seen most clearly once the hairs have been removed, the
hairs from the donor region are removed by plucking hair out
of the donor region and selecting the suitable hairs in the
anagen phase. Tweezers are, for example, very suitable for plucking
hair from the donor region.
In step (2) of the method according to the invention the hairs
are cultured from the hair removed, under conditions such that
the hair follicle cells are able to multiply. In principle,
the hair follicle cells can be cultured from the hairs by the
known method. Culture media for this purpose are available commercially
and any associated growth supplements are freely available.
Such culture media are also termed serum-free keratinocyte culture
media and usually contain essential and non-essential amino
acids, vitamins, trace elements, organic constituents and inorganic
salts. Such culture media can also be combined with growth supplements,
which contain growth factors, hormones, antibiotics and tissue
extracts, such as, for example, BPE (Bovine Pituitary Extract),
insulin, hydrocortisone, HEGF (Human Epidermal Growth Factor),
TGF (Transformal Growth Factor), L-cysteine, L-leucine and gentamicin.
According to the invention, particularly good results are obtained
if, in step (2) of the method according to the invention, use
is made of a culture medium which, in addition to the known
culture media described above, is optionally supplemented with
one or more growth supplements, and/or is supplemented with
one human mast cell line and/or autologous (cultured) CD34.sup.+
cells (CD34.sup.+ positive cells) or one or more extracts of
the human mast cell line(s) and/or of the CD34.sup.+ cells and/or
growth-stimulating agents. Preferably, only cultured CD34.sup.+
cells are used because these cells are native to the body. Human
mast cells are known per se They contain various growth factors
and are produced and consumed in various different sites in
the body. The extracts of the human mast cell line and/or autologous
(cultured) CD34.sup.+ cells and/or growth-stimulating agents
contain growth factors. However, to date such extracts of the
human mast cell line and/or autologous (cultured) CD34.sup.+
cells and/or growth-stimulating agents have not been used in
media for culturing hair follicle cells. A human mast cell line
from which the extracts can be used, is HMC-1 (Human Mast Cell
line 1) or a cell line derived therefrom, such as the subclones
5C6 and KU812. Mast cell lines such as HMC-1 and subclones thereof
are commercially available. According to the invention, instead
of mast cells, autologous (cultured) CD34.sup.+ cells or a combination
of two or more of these cell types can be used.
The extracts of the human mast cell line and/or autologous (cultured)
CD34.sup.+ cells and/or growth-stimulating agents can very suitably
be introduced into the medium by adding at least one degranulating
agent to the serum-free keratinocyte culture medium containing
the human mast cell line, a subclone thereof and/or autologous
(cultured) CD34.sup.+ cells and/or growth agents. An inorganic
salt can also be added to optimise the action of the degranulating
agent. A suitable salt in this connection is CaCl.sub.2. The
degranulating agent then, as it were, cuts the mast cells and/or
the autologous (cultured) CD34.sup.+ cells into smaller pieces
(degranulation) as a result of which the growth factors are
released into the medium. After degranulation, the residues
of the mast cells and/or the autologous (cultured) CD34.sup.+
cells can be removed, for example by means of centrifuging.
The resultant, conditioned medium can then be used to culture
the hair follicle cells.
Degranulating agents are known per se and can roughly be divided
into three groups:
hormones, such oestrogens and bradykinin,
neurotransmitters and antigens, such as apitoxin. Substance
P and Ig E, and
synthetic agents, such as Compound 48/80 and Ionophore A23187.
Those degranulating agents which are capable of degranulating
the mast cells optionally used are suitable for use in the method
according to the invention.
An alternative is first separately to prepare the extracts or
the human mast cell line and/or autologous (cultured) CD34.sup.+
cells and/or growth agents and to add these extracts as such
to the serum-free keratinocyte culture medium.
The conditions under which the hair follicle cells are cultured
in step (2) can vary substantially and are dependent on, inter
alia, the medium used. However, it has been found that the culturing
of the hair follicle cells at a temperature of 30-40.degree.
C., preferably in an incubator so that the temperature can be
kept constant, in an atmosphere which is saturated with water
and which contains 3-10% by volume CO.sub.2 in addition to the
other customary constituents of air in the customary quantities
gives good results. However, these conditions are in no way
restrictive and other conditions can therefore also be used.
The culture period varies per culture, usually from 1 hour to
40 days. In order to obtain a good result, it is preferable
to replace the medium by fresh medium every 2 to 5 days.
Step (2) of the method according to the invention can be carried
out in a single culture step, but can also be carried out in
several sub-steps by culturing subcultures from the initial
culture or cultures. This can be carried out by taking a sufficiently
large number of hair follicle cells from the main culture when
the latter contains sufficient hair follicle cells. The hair
follicle cells from the main culture are cultured in turn, preferably
--but not necessarily-- in the same culture medium as that in
which the original main culture was cultured. In the course
of time it is possible, if desired, to culture yet further cultures
from said subcultures, etc. etc.
After the hair follicle cells have been cultured they must be
detached from the substrate on which they have been grown. This
can be carried out by the known methods, for example by making
use of a trypsin solution (0.1-0.5% by weight in water), optionally
in combination with an EDTA solution (0.01-0.05% by weight)
in PBS (Phosphate Buffered Saline). Such methods are known per
se. Before the cultured hair follicle cells are used in step
(3), the most cells and/or autologous (cultured) CD34.sup.+
cells can, if desired, be removed.
In step (3) or the method according to the invention, the cultured
hair follicle cells and/or autologous (cultured) CD34.sup.+
cells are implanted in the skin in the receptor region. It has
been found that very good results are obtained when the cultured
hair follicle cells and/or autologous (cultured) CD34.sup.+
cells are implanted directly into the pores of the receptor
region. If the pores are difficult to see, which can with a
pale skin colour in particular, the pores can be made more readily
visible by applying a (usualy yellow-coloured) liquid to the
skin in the receptor region, which becomes darker in colour
in the positions where the pores are located as a result of
the secretion of the perspiration from the sweat glands present
in the pores. An example or such a liquid is a 1-10% solution
of o-phthaldialdehyde in, for example xylene or ethyl ether,
as described by L. Juhlin and W. B. Suelley in Nature, Jan.
28, 1967, page 408.
Preferably a quantity of a suspension of cultured hair follicle
cells and/or autologous (cultured) CD34.sup.+ cells is introduced
into each pore such that said quantity contains sufficient hair
follicle cells and/or autologous (cultured) CD34.sup.+ cells
to allow a hair follicle to develop from which a hair is able
to grow. The number of cultured hair follicle cells and/or autologous
(cultured) CD34.sup.+, cells needed to cause a hair follicle
to form is highly dependent on the degree of differentiation
or the growth potential of the cultured cells. Naturally, fewer
cultured cells will be needed when the cultured hair follicle
cells and/or autologous (cultured) CD34.sup.+ cells have a relatively
high degree of differentiation than when the cultured cells
have a relatively low degree of differentiation. There are a
large number of factors which influence the degree of differentiation
of the cultured hair follicle cells and/or autologous (cultured)
CD34.sup.+ cells. For example, the medium used and the culture
time are factors which play a role. Preferably, a suspension
of the cultured hair follicle cells and/or autologous (cultured)
CD34.sup.+ cells in the medium in which said cells were cultured
is used. The suspension preferably has a hair follicle cell
concentration which is such that 0.015-5 .mu.l, preferably 0.1
to 0.5 .mu.l, of suspension per pore can suffice. A small quantity
such as this is preferably injected into the pore with the aid
of a metering device which is provided with a hollow needle.
preferably an 18 to 40 Gauche (G) needle. The metering device
used can be, for example, an electronic metering system for
a pipette, a 18-40 G needle being fitted on the end of the metering
hose or of the pipette. In the light of the average size of
the pores in, in particular, the scalp, it is preferable to
use a hollow 34 G needle. However, smaller or larger needles
can also be used on other parts of the body. It is also possible
and preferable for the suspension to be injected with an (electronic)
repeating injection-metering apparatus such as an insulin pen.
In that case, a needle specially developed for the purpose,
that is to say a "follicle-conducting needle", is also used
to introduce the cells at the desired position and to damage
the skin as little as possible. A follicle-conducting needle
is a needle which is flexible and minuscule and also which the
hair follicle can follow.
During the injection of the suspension, substances can also
be introduced at the same time which keep the cells alive for
longer. Examples of such substances are Minoxidil and other
growth-stimulating agents.
The invention also relates to a method for culturing hair follicle
cells from hairs in the anagen phase, one or more hairs in the
anagen phase being placed in a medium which is supplemented
with one suitable serum-free keratinocyte culture medium and,
optionally, supplemented with one human mast cell line and/or
autologous (cultured) CD34.sup.+ cells or one or more extracts
of the human mast cell line(s) and/or of the CD34.sup.+ cells
and/or growth-stimulating agents. The way in which such a medium
can be obtained has already been described above.
As far as the preferences with regard to the various constituents
of the medium to be used and to the culturing conditions are
concerned, these are the same as those for the medium and the
conditions employed in step (2) of the method already described
above. Therefore, it is preferable to use one mast cell line,
a subclone thereof and/or autologous (cultured) CD34.sup.+ cells
and/or growth-stimulating agents. Suitable conditions comprise
a culture temperature of 30-40.degree. C. in an atmosphere which
is saturated with water and contains 3-10% by volume of CO.sub.2.
The invention also relates to a medium for culturing hair follicle
cells from hairs it the anagen phase and to a method for the
preparation of said medium. The medium according to the invention
comprises at least one suitable serum-free keratinocyte culture
medium, optionally supplemented with one human mast cell line
and/or autologous (cultured) CD34.sup.+ cells or extracts of
the human mast cell line(s) and/or of the CD34.sup.+ cells and/or
growth-stimulating agents. The special feature of such a medium
is, in particular, that it contains growth factors originating
from one or more human mast cell lines and/or autologous (cultured)
CD34.sup.+ cells.
A preferred medium comprises a serum-free keratinocyte culture
medium and extracts of the human most cell line HMC-1 or a cell
line derived therefrom, such as 5C6 or KU812 or autologous (cultured)
CD34.sup.+ cells. Further details on the constituents of the
medium according to the invention have already been given above.
The method for the preparation of said medium comprises the
following steps:
(a) addition of at least one mast cell line, subclone thereof
and/or autologous (cultured) CD34.sup.+ cells and/or growth-stimulating
agents and optionally one degranulating agent, optionally in
combination with an organic salt, such as, for example, CaCl.sub.2,
to a serum-free keratinocyte culture medium;
(b) degranulation of the mast cell line(s), subclones thereof
and/or autologous (cultured) CD34.sup.+ cells, so that the growth
factors are released into the medium; and
(c) optionally removal of the mast cells and/or autologous (cultured)
CD34.sup.+ cells from the medium, for example by centrifuging,
after which the (conditioned) medium is obtained.
According to the invention, the mast cells and/or the CD34.sup.+
cells and/or the residues thereof are removed from the medium.
The conditions under which this method can be carried out are
not particularly critical and will be obvious to any person
skilled in the art. The degranulating agent used in step (a)
is preferably Compound 48/80 in combination with CaCl.sub.2.
Finally, the invention relates to a precursor medium for culturing
hair follicle cells from hairs in the anagen phase which medium
comprises at least one suitable serum-free keratinocyte culture
medium and which is optionally supplemented with one human mast
cell line and/or autologous (cultured) CD34.sup.+ cells or one
or more extracts of the human mast cell line(s) and/or or the
CD34.sup.+ cells and/or growth-stimulating agents and/or at
least one degranulating agent. Said precursor medium can be
used as such for preparing therefrom a suitably conditioned
medium for culturing hair follicle cells from hairs in the anagen
phase. This can be performed simply by carrying out the degranulation
if both a human mast cell line and/or autologous (cultured)
CD34.sup.+ cells is/are present as well as degranulating agent.
If one of the two is absent, that is to say the mast cells and/or
the CD34.sup.+ cells and the degranulating agent, respectively,
the missing component must be first added before degranulation
of the mast cells can be carried out.
* * * * *
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