U.S. patent application number 09/091483 was filed with the patent office on 2002-01-31 for improved immortalized human skin cell lines and novel serum-free medium useful for the production thereof.
Invention is credited to BAUR, MARKUS, MACE, CATHERINE, MALNOE, ARMAND, PFEIFER, ANDREA, REGNIER, MARCELLE.
Application Number | 20020012993 09/091483 |
Document ID | / |
Family ID | 24304605 |
Filed Date | 2002-01-31 |
United States Patent
Application |
20020012993 |
Kind Code |
A1 |
BAUR, MARKUS ; et
al. |
January 31, 2002 |
IMPROVED IMMORTALIZED HUMAN SKIN CELL LINES AND NOVEL SERUM-FREE
MEDIUM USEFUL FOR THE PRODUCTION THEREOF
Abstract
The present invention relates to improved continuous
(immortalized) cell lines, in particular keratinocytes and
melanocytes derived from normal human skin tissue. The present
invention also relates to novel serum-free media for isolating,
producing and maintaining said improved continuous keratinocyte and
melanocyte cell lines. The present invention also relates to
methods for producing primary melanocytes and keratinocytes under
serum-free conditions without any feeder cells.
Inventors: |
BAUR, MARKUS; (LAUSANNE,
CH) ; MACE, CATHERINE; (LUTRY, CH) ; MALNOE,
ARMAND; (DOMMARTIN, CH) ; PFEIFER, ANDREA;
(ST-LEGIER, CH) ; REGNIER, MARCELLE; (PARIS,
FR) |
Correspondence
Address: |
WINSTON & STRAWN
200 Park Avenue
New York
NY
10166-4193
US
|
Family ID: |
24304605 |
Appl. No.: |
09/091483 |
Filed: |
June 19, 1998 |
PCT Filed: |
December 19, 1996 |
PCT NO: |
PCT/EP96/05812 |
Current U.S.
Class: |
435/371 ;
435/404 |
Current CPC
Class: |
C12N 2500/20 20130101;
C12N 2500/44 20130101; C12N 2500/25 20130101; C12N 5/0629 20130101;
C12N 2500/38 20130101; C12N 2500/40 20130101; C12N 2501/39
20130101; A61K 35/12 20130101; C12N 2500/90 20130101; C12N 2501/115
20130101; C12N 2500/46 20130101; C12N 2501/81 20130101; C12N
2501/11 20130101; C12N 2501/999 20130101; C12N 2500/32 20130101;
C12N 2510/04 20130101 |
Class at
Publication: |
435/371 ;
435/404 |
International
Class: |
C12N 005/08; C12N
005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 1995 |
US |
08/576483 |
Claims
What is claimed is:
1. An immortalized human keratinocyte cell line or melanocyte cell
line which retains the ability to differentiate and to express
proteins and enzymes which are expressed by normal differentiated
keratinocytes or melanocytes even after high passage in tissue
culture.
2. An immortalized keratinocyte cell line according to claim 1
wherein said cell line expresses keratin proteins and other
proteins which are expressed by normal differentiated
keratinocytes.
3. An immortalized keratinocyte cell line according to claim 2
wherein said keratin proteins include Keratin K1/10, Keratin K14
and said other proteins include involucrin, filaggrin and
loricrin.
4. An immortalized keratinocyte cell line according to claim 1
wherein said immortalized keratinocyte cell line exhibits a CYP450
profile which is identical or substantially identical to that of
normal differentiated keratinocytes.
5. An immortalized keratinocyte cell line according to claim 4
wherein said cell line expresses CYP450 1A1, 2C, 2E1 and 3A5 but
does not express CYP450 1A2, 2A6, 2B6 and 2D6.
6. An immortalized human keratinocyte cell line according to claim
1 which is produced under serum-free conditions without the use of
feeder cells.
7. An immortalized human melanocyte cell line according to claim 1
which is produced under serum-free conditions without the use of
feeder cells.
8. An SV40 T-Antigen immortalized human skin cell line selected
from the group consisting of the keratinocyte lines DK2-NR (DSM
ACC2238), DK3-NR (DSM ACC2239) and FK2-NR (DSM ACC2240) and the
melanocyte line DM2-NR (CNCM I-1796).
9. An immortalized keratinocyte cell line according to claim 1
which expresses mRNA encoding glutathione-S-transferase GST-.pi.,
GST-.mu. and GST-.alpha..
10. An immortalized keratinocyte cell line according to claim 1
which when cultured in organotypic culture formns a highly
stratified and polarized epithelium having cornified superficial
layers in the absence of serum or feeder cells.
11. An immortalized keratinocyte cell line according to claim 1
which expresses collagenase type 1 and TNF-.alpha. when treated
with phorbol esters.
12. An improved method for immortalizing human skin cells to obtain
immortalized keratinocytes and melanocytes comprising the following
steps: (i) obtaining a human skin tissue sample; (ii) preparing
said skin sample for culturing in vitro; (iii) obtaining
keratinocytes and/or melanocytes from said prepared skin sample and
seeding said keratinocytes and/or melanocytes into a serum-free
growth medium, onto culture plates provided with a coating
comprising fibronectin, type 1 collagen and BSA which facilitates
cell attachment and growth; (iv) changing the medium as necessary
to optimize confluent growth of the cultured cells while
continuously maintaining the coating on the culture plates; (v)
transferring the keratinocytes or melanocytes into a serum-free
selection medium onto similarly precoated culture plates; (vi)
infecting the keratinocytes or melanocytes with a retroviral
construct; (vii) transferring the resultant immortalized
keratinocytes or melanocytes to a serum-free proliferation medium
suitable for proliferating immortalized keratinocytes or
melanocytes onto similarly precoated culture plates; and (viii)
transferring the resultant proliferated keratinocytes to a
serum-free differentiation medium, which contains high calcium onto
similarly precoated culture dishes.
13. The method of claim 12 wherein the retroviral construct is the
SV40 construct pLXSHD+SV40(#328) or the HPV16 construct
pLXSHD+E6/E7.
14. The method of the claim 12 wherein the serum-free medium in
step (iii) is NR-3 medium.
15. The method of claim 12 wherein the medium in step (v) is the
NR-3 medium or the NR-4 medium.
16. The method of claim 12 wherein the proliferation medium in step
(vii) is the NR-2 medium or the NR-3 medium and M2 medium for
melanocytes.
17. The method of claim 12 wherein the differentiation medium in
step (viii) is the NR-2 medium or modified MCDB 153 medium which
contains a calcium content of at least 1.5 mM.
18. A novel serum-free culture medium suitable for isolating and
producing human keratinocytes and melanocytes which retain the
ability to differentiate and to express proteins and enzymes of
differentiated human keratinocytes and melanocytes even after high
passage which comprises: amino acids or amino acid salts; inorganic
salts; vitamins; adenine, ethanolamine, glucose, HEPES, phenol red,
putrescine 2HCl, thioctic acid, thiamine HCl, thymidine, epidermal
growth factor; insulin; hydrocortisone; transferrin;
phosphoethanolamine; and bovine pituitary extract.
19. The medium of claim 18 which further contains epinephrine.
20. The medium of claim 19 wherein the epinephrine concentration is
sufficient to enhance growth of keratinocytes.
21. The medium of claim 18 wherein the amino acids contained in the
medium are selected from the group consisting of L-alanine,
L-arginine-HCl, asparagine-H.sub.2O, L-aspartic acid,
L-cysteine-HCl-H.sub.2O, L-glutamic acid, glutamine, glycine,
L-histidine-HCl-H.sub.2O, L-isoleucine, L-leucine, L-lysine-HCl,
L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,
L-tryptophan, L-tyrosine, L-valine and salts thereof.
22. The medium of claim 18 wherein said inorganic salts are
selected from the group consisting of ammonium metavanadate,
ammonium molybdate, calcium chloride, cupric sulfate, ferric
sulfate, magnesium chloride, manganese chloride, nickel sulfate,
potassium chloride, sodium acetate, sodium bicarbonate, sodium
chloride, sodium phosphate dibasic, sodium pyruvate, sodium
selenite, sodium silicate, tin chloride and zinc sulfate.
23. The medium of claim 18 wherein the vitamins are selected from
the group consisting of d-biotin, d-calcium pantothenate, choline
chloride, cyanocobalamin, folic acid, i-inositol, nicotinamide,
pyridoxine and riboflavin.
24. A novel serum-free medium for isolating, producing and/or
maintaining immortalized keratinocytes and/or melanocytes which is
selected from the group consisting of the NR-2 medium, the NR-3
medium and the NR-4 medium.
25. An improved assay which uses differentiated keratinocytes
and/or melanocytes wherein the improvement comprises using
immortalized keratinocytes and/or melanocytes according to claim
1.
26. The assay of claim 25 wherein the cells are selected from the
group consisting of DK2-NR (DSM ACC2238), DK3-NR (DSM ACC2239),
DM2-NR (CNCM I-1796) and FK2-NR (DSM ACC2240).
27. The method of claim 24 wherein said assay is an inflammation
assay.
28. An improved assay which uses primary melanocytes or
keratinocytes wherein the improvement comprises using primary
melanocytes or keratinocytes obtained according to claim 12
(iii).
29. The assay of claim 28 wherein said assay is an inflammation
assay.
30. An improved skin grafting method wherein the improvement
comprises using as the grafted skin tissue primary keratinocytes or
melanocytes produced according to claim 12 (iii).
Description
[0001] The present invention relates to improved continuous
(immortalized) cell lines derived from normal human skin tissues,
in particular keratinocytes and melanocytes which retain the
ability to express differentiation proteins characteristic of
differentiated melanocytes or keratinocytes, even in high passages.
The present invention also relates to novel serum free media which
do not require the use of feeder cells.
BACKGROUND OF THE INVENTION
[0002] The production of immortalized cell lines derived from human
skin tissues has been previously described. In general such methods
comprise transfection or transformation of human skin cells, e.g.,
keratinocytes and melanocytes, cultured in vitro with agents which
provide for immortalization.
[0003] Immortalization refers to the production of cells which are
able to be cultured for prolonged time periods in vitro, ideally
indefinitely. These cells are also referred to as continuous cell
lines. By contrast non-immortalized cells are only capable of
growing for a finite number of cell divisions in vitro.
Immortalized cells are highly desirable because they provide a
stable, potentially infinite supply of cells having defined
characteristics. Conventional agents for producing immortalized
cell lines and immortalized human skin cell lines in particular
include, e.g., viruses, recombinant viruses, and plasmids which
contain DNA sequences which provide for immortalization.
[0004] Probably the most common method of producing immortalized
human cell lines involves the use of SV40 sequences and more
specifically the SV40 large T antigen DNA as an immortalizing
agent. For example, Steinberg et al., J. Cell Phys., 123:117-125
(1985); Reddel et al., U.S. Pat. No. 4,885,238 issued on Dec. 5,
1989; Major, U.S. Pat. No. 4,707,448 issued on Nov. 17, 1987;
Stoner et al., Cancer Res., 51:365-371 (1991); Chopra et al., In
Vitro Cell Dev. Biol., 30A:539-546 (1994); Chopra et al., In Vitro
Cell Dev. Biol., 27A:763-765 (1991); Christian et al., Cancer Res.,
47:6066-6073 (1987); Rhim et al., Science, 227:1250-1252 (1985);
and Grubman et al., Gastrointest. Liver Physiol., 29:G1060-G1070
(1994) teach the use of SV40 vectors and SV40 large T antigen
sequence containing vectors to produce immortalized human cell
lines. The introduction of such sequences is generally effected by
infection using SV40 virus or with a hybrid adenovirus-12 SV40
hybrid virus or by transfection of cells with a recombinant plasmid
containing the Rous sarcoma virus long terminal repeat and the
Ori-SV40 early region by strontium phosphate co-precipitation. (See
Brash et al., Mol. Cell Biol., 7:2031-2034, (1987)).
[0005] Another known method for producing immortalized cell lines,
and immortalized human keratinocytes in particular, involves
transfection or infection of cells with human papillomavirus DNA
sequences. For example, U.S. Pat. No. 5,376,542 by Schlegel issued
on Dec. 27, 1994 describes immortalization of human epithelial
cells with isolated HPV-16, 18, 31, 33 or 35 E6 and E7 genes or the
E7 gene alone to produce non-tumorigenic immortalized cell lines.
Also, Barbosa et al., Oncogene, 4:1529-1532 (1989); and Munger et
al., J. Virol., 63(10):4417-4421 (1989) teach the use of HPV-16 and
HPV-18 E6 and E7 genes to produce immortalized human
keratinocytes.
[0006] However, while numerous groups have reported immortalized
keratinocyte cell lines, and their usage in in vitro assays,
previous immortalized keratinocyte cell lines and melanocyte cell
lines have typically exhibited one or more properties which render
their usage disadvantageous. For example, previously reported
immortalized keratinocytes have exhibited one or more of the
following characteristics: (i) reduction or loss of expression of
differentiation markers, e.g., proteins which are expressed by
normal differentiated keratinocytes, and (ii) altered growth
characteristics in tissue culture.
[0007] For example, Jetten et al, J. Invest. Dermatol., 92:203-209
(1989) reports SV40 immortalized keratinocytes obtained after high
passage number (>passage 12) using the vector NHEK-SV40-T8-1
which are unable to differentiate. Similarly, Bernard et al, Cancer
Res., 45:1707-1716 (1985) report isolation of an immortalized
keratinocyte cell line referred to as SVK14 which is reported to be
almost completely unable to differentiate. Also, this cell line
shows no expression of keratins K1/10 (>53 kD) and the 50 kD
keratin (keratin K14), which proteins are normally expressed by
differentiated keratinocytes.
[0008] Still further, Steinberg et al, J. Cell. Physiol.,
123:117-125 (1985) report SV40 transformed keratinocytes which
gradually lose the ability to express keratins which are
characteristic of the normal keratin cytoskeleton. This loss of
normal keratin expression occurs after about 10-15 passages. Also,
Hronis et al, Cancer Res., 44:5797-5804 (1984) teach SV40 DNA
immortalized keratinocytes which have lost the ability to produce
K5, K6, K14/15, K16 and K17 keratins and involucrin which proteins
are characteristic of normal differential keratinocytes. Still
further, Morris et al, Proc. Natl. Acad. Sci. USA, 82:8498-8502
(1985) teach SV40 immortalized keratinocytes which at higher
passages (>passage 14) exhibit highly reduced expression of
Class II and Class I keratins. For example, these keratinocytes
exhibit almost no expression of K13 (Id.).
[0009] Also, Banks-Schlegel et al, J. Cell. Biol., 96:330-337
(1983) disclose SV40 immortalized keratinocytes which exhibit
altered growth characteristics in tissue culture. For example,
unlike normal keratinocytes, these immortalized cells require a 3T3
feeder layer to grow.
[0010] Previously described methods for producing immortalized
human keratinocytes and melanocytes have typically used the feeder
cell technique (wherein fibroblasts usually function as the
"feeder" cells) and generally culture cells in serum-containing
medium. For example, Sexton et al., "Stable transfection of human
keratinocytes: HPV immortalization," Keratinocyte Methods, eds.,
Leigh, I. M. et al., University Press, 179-180, (1994); Garlick,
"Retroviral Vectors," Keratinocyte methods, (eds. Leigh I. M. et
al., Cambridge University Press, 181-183 (1994)) teach the use of
fetal calf serum containing medium and feeder cells in the
isolation and production of immortalized keratinocytes.
[0011] The use of serum-free medium during the isolation and
production of immortalized epithelial cells, and specifically human
keratinocytes has been previously described. For example, Barbosa
et al., Oncogene, 4:1529-1532 (1989) describe initially culturing
human keratinocytes transfected by electroporation or lipofection
in low calcium, serum-free medium until confluence.
[0012] However, notwithstanding what has been previously reported,
there still exists a significant need in the art for immortalized
human keratinocytes and melanocytes which possess improved
properties, in particular which maintain the differentiation
potential of normal keratinocytes and melanocytes and which express
differentiation proteins characteristic of differentiated
melanocytes and keratinocytes, even after high passages. Such cells
would be highly beneficial for many usages, in particular in assays
which require differentiated skin cells. There further exists a
need in the art for improved culture media capable of maintaining
primary and immortalized keratinocytes and melanocytes, as well as
improved culturing methods which do not require the use of feeder
cells.
OBJECTS OF THE INVENTION
[0013] Towards that end, it is an object of the invention to
produce improved continuous (immortalized) cell lines derived from
normal human skin tissue, especially immortalized keratinocytes
and/or melanocyte cell lines derived from normal human skin tissue
which maintain the ability to differentiate and to express
differentiation proteins even after high passages. More
specifically, it is an object of the invention to obtain
immortalized keratinocytes which maintain the ability to express
keratins, cytochromes, as well as other differentiation proteins,
e.g., involucrin, filaggrin and loricrin, which are either not
expressed or poorly expressed by conventional immortalized
keratinocyte cell lines. It is still another object of the
invention to obtain immortalized keratinocytes and melanocytes
which express enzymes which are normally expressed by
differentiated keratinocytes and melanocytes, especially phase II
enzymes such as glutathione-S-transferases as well as enzymes
and/or proteins which are involved in cellular oxidation and
inflammatory responses.
[0014] It is another object of the invention to provide novel serum
free media for culturing, producing and maintaining normal or
continuous keratinocytes and/or melanocytes in tissue culture.
These novel serum free media are also useful for isolating,
establishing and immortalizing human skin cells for obtaining
continuous melanocytes and keratinocytes according to the
invention. It is thus a specific object of the invention to provide
a fully defined culture medium (without unknown or ill-defined
supplements) for culturing keratinocytes without feeder cells
containing epinephrine which has surprisingly been discovered to be
a strong growth potentiator of normal keratinocytes in serum-free
medium.
[0015] It is another object of the invention to provide a novel
method for isolating, establishing and immortalizing human skin
cells for obtaining continuous melanocyte and keratinocyte cell
lines derived from normal skin tissue, said method using serum-free
media, notably those according to the invention, and a cell
attachment cocktail containing fibronectin, BSA and type I collagen
without "feeder cells" (e.g., fibroblasts).
[0016] It is another object of the invention to provide primary
keratinocyte or melanocytes produced under serum free conditions
without the use of any feeder cells, said primary keratinocytes and
melanocytes being used for skin grafting and in ex vivo genetic
therapy.
[0017] It is another object of the invention to provide methods of
using such novel and improved continuous keratinocyte and
melanocyte cell lines, e.g., for immunological, pharmacological,
photo- and chemotoxicological skin reaction assays and for the
expression of heterologous genes.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 compares the growth (in terms of cell number) of
non-immortalized DKO-NR keratinocytes in three different media,
i.e., NR-3, epinephrine-supplemented modified MCDB 153 and MCDB 153
after 6 days.
[0019] FIG. 2a depicts the SV40 retroviral construct
pLXSHD+SV40(#328) preferably used to immortalize the subject
melanocytes and/or keratinocytes
[0020] FIG. 2b depicts the HPV16 retroviral construct
pLXSHD+E6/E7.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention provides continuous (immortalized)
cell lines derived from normal human skin tissues, i.e.,
immortalized keratinocytes and melanocytes, which maintain the
ability to differentiate and which maintain the ability to express
differentiation proteins which are expressed by normal
keratinocytes or melanocytes even in high passages. By high passage
is meant at least 10 passages in culture, preferably at least 20-30
passages, more preferably for at least 50 passages, and ideally for
an infinite number of passages. For example, immortalized
keratinocytes produced according to the invention express the
differentiation proteins keratin K1/10, keratin K14, involucrin,
filaggrin and loricrin even after high passages in tissue culture.
This is in contrast to previously reported immortalized
keratinocytes which either do not express these differentiation
proteins or which express these differentiation proteins
poorly.
[0022] Also, the present invention provides primary keratinocytes
and melanocytes produced under serum free conditions and without
feeder cells which maintain the ability to differentiate and to
express proteins characteristic of differentiated melanocytes and
keratinocytes.
[0023] The subject immortalized keratinocytes have a cytochrome
p450 profile (CYP450) which is similar if not identical to that of
normal keratinocytes. For example, the subject cells express CYP450
3A5 and not CYP450 3A4. Also, the subject immortalized
keratinocytes express phase II enzymes, e.g.,
glutathione-S-transferase (GST) and more specifically GST.alpha.,
GST.mu. and GST.pi. comparably to normal non-immortalized
keratinocytes.
[0024] Further, the subject immortalized keratinocytes express
proteins and enzymes involved in the cellular oxidation and
inflammatory responses, e.g., superoxide dismutase (SOD), and type
I collagenase and tumor necrosis factor alpha (TNF.alpha.) after
treatment with phorbol esters similarly or identically to normal
differentiated keratinocytes. Given these characteristics, these
cell lines provide a highly stable, reproducible source of cells
for immunological, pharmacological, inflammation, photo- and
chemotoxicological skin reaction studies.
[0025] Further, the subject immortalized melanocytes express
endogenously melanin associated proteins (see examples 14-15
hereafter).
[0026] Also, the subject immortalized keratinocyte cell lines and
melanocyte cell lines when cultivated in organotypic culture form a
highly stratified and polarized epithelium having cornified
superficial layers (stratum corneum). This had only been previously
achieved under conventional culture conditions, i.e., medium
containing serum, feeder layer technique (See, e.g., Lechner et al,
Virology, 185:536-571, 1991).
[0027] Further, the subject immortalized keratinocytes and
melanocytes are obtained from normal skin under serum-free
conditions and without the use of any feeder cells. In general, the
subject immortalized keratinocytes and melanocytes are obtained by
the following steps:
[0028] (i) obtaining a human skin tissue sample;
[0029] (ii) preparing said skin sample for culturing in vitro;
[0030] (iii) obtaining keratinocytes and/or melanocytes from said
prepared skin sample and seeding said keratinocytes and/or
melanocytes into a serum-free growth medium, preferably either the
NR-3 medium or NR-4 (for melanocytes) (described infra) onto
culture plates provided with a coating which facilitates cell
attachment and cell growth, said coating comprising fibronectin,
type 1 collagen and BSA.
[0031] (iv) changing the medium as necessary to optimize confluent
growth of the cultured cells while continuously maintaining the
coating on the culture plates;
[0032] (v) transferring the keratinocytes or melanocytes into an
selection medium, preferably a serum-free medium, onto similarly
precoated culture plates;
[0033] (vi) infecting the keratinocytes or melanocytes with a
retroviral construct, preferably a SV40 or papilloma virus 16 based
construct, such as the SV40 plasmid pLXSHD+SV40(#328) which
contains the large T antigen (T Ag) of the Simian virus 40, or the
plasmid pLXSHD+E6/E7 which contains the E6/E7 gene of the papilloma
virus 16 (HPV16) (described infra);
[0034] (vii) transferring the resultant immortalized keratinocytes
or melanocytes to a proliferation medium suitable for proliferating
immortalized keratinocytes or melanocytes onto similarly precoated
culture plates, preferably NR-2 or NR-3 medium (described infra)
and the melanocyte medium M2 (source M. Olsson, Inst. of
Dermatology, Uppsala, Sweden); and
[0035] (viii) transferring the resultant proliferated keratinocytes
to a differentiation medium, preferably NR-2 (described infra) or
modified MCDB 153 medium (described infra) which contains high
calcium, preferably 1.5 mM onto similarly precoated culture disks
(Boyce et al., J. Tissue Cult. Meth., 9:83-93, 1985; Pittlekow et
al., J. Invest. Dermatol., 86:410-417, 1986).
[0036] More specifically, step (i) will typically comprise
obtaining human skin tissue samples from human donors, e.g., those
obtained during surgery or pediatry. The immortalization of a
single skin cell sample, i.e. autologous skin cell sample, allows
for the production of immortalized keratinocyte and melanocyte cell
lines which exhibit defined characteristics, e.g. a particular
receptor profile which is characteristic of a particular donor.
[0037] The skin sample will then be prepared in step (ii) such that
it is suitable for culturing in vitro. This will preferably be
effected by initially washing the skin sample, e.g. using the same
medium which is used for culturing. Preferably this is effected in
NR-2 medium, a serum-free medium, the exact composition of which is
disclosed infra, which has been found to be advantageous for
culturing normal keratinocytes and melanocytes. After washing, the
skin sample will then preferably be shaved, e.g. with a dermatome,
and then excised into small pieces.
[0038] The resultant skin sections are then preferably separated
into dermis and epidermis. This may be effected by physical and/or
enzymatic means. For example, this may be effected by
trypsinization, e.g. by floating skin sheets in a trypsin solution
(e.g. about 0.5%) containing EDTA (e.g. about 0.1%) for a
sufficient time to effect cell separation, e.g. about 30-60 minutes
at 37.degree. C. or overnight at 4.degree. C.
[0039] The dermis is separated (to isolate the fibroblasts, see
EXAMPLE 2) and the epidermis is then placed in a suspension medium.
Preferably the suspension medium will contain soybean trypsin
inhibitor solution (SBTI) and will be contacted with the cells for
a sufficient time, typically about 5 minutes, in order to
inactivate the trypsin and provide for cell release. Tissue culture
medium will then be added, preferably serum-free NR-2 medium
(described infra) and a filter (e.g. 100 mm filter) to obtain the
desired cells, i.e. keratinocytes and/or melanocytes.
[0040] The resultant primary keratinocyte/melanocyte culture
obtained in step (ii) is then seeded into serum-free medium,
preferably NR-3 medium (described in detail infra), at a suitable
cell concentration, preferably about 1.2.times.10.sup.4
cells/cm.sup.2, onto precoated culture plates. However, this cell
concentration may be varied within wide limits. The culture plates
are preferably continuously coated with a composition which has
been surprisingly found to enhance both the attachment and growth
of keratinocytes and melanocytes, specifically a solution of
fibronectin, BSA and collagen type 1. This cell coating composition
has previously been described for use with bronchial cells.
(Lechner et al, J. Tissue Cult. Meth. 9:43-48 (1985)), which
reference is incorporated by reference herein.
[0041] In step (iv) the culture medium is changed as often as
necessary to optimize cell growth. Preferably, the medium will be
changed about every second day. However, this may vary dependent
upon the particular skin sample. After reaching almost total
confluence, e.g. about 90% confluence, which typically occurs after
about 10-14 days, the keratinocytes and melanocytes are then
separated. This may be accomplished by any means which provides for
adequate cell separation without adversely affecting the
melanocytes and/or keratinocytes. For example, this may be effected
by differential trypsinization. Preferably, the melanocytes or
keratinocytes will be treated with a trypsin/EDTA solution, and
then transferred to the selection medium. In the case of
keratinocytes, the cells are preferably treated for about 5-10
minutes with trypsin/EDTA solution (0.025%/0.01%) and then in step
(v) seeded into NR-3 medium onto precoated plates. It is important
to note that NR-3 medium promotes the growth of keratinocytes
versus melanocytes. In the case of melanocytes, the cells are
preferably treated for about 2-4 minutes with trypsin/EDTA
(0.025%/0.01%) and then seeded in step (v) into NR-4 medium onto
similarly precoated culture plates. It is important to note that
NR-4 medium specifically inhibits the growth of keratinocytes.
[0042] These cells will then be treated with the immortalizing
agent. Alternatively, the cells may be frozen until immortalization
is to be effected, e.g. in liquid N.sub.2. Infection and
immortalization is preferably effected using the SV40 construct
identified as pLXSHD+SV40(#328) which is depicted in FIG. 2a and
which was described by Stockshlaeder et al (GeneBank, n.sup.o
accession M64753; Stockshlaeder et al., Human Gen. Therapy, 2,
33-39, 1991), or the HPV16 construct identified as pLXSHD+E6/E7
which is depicted in FIG. 2b. The pLXSHD+SV40(#328) construct
contains the SV40 T-Ag sequence, 5' and 3' LTR sequences of SV40,
pBR322 sequences which provide for replication in E. coli, a
multiple cloning site, an SV40 polyadenylation sequence, among
other sequences. The pLXSHD+E6/E7 construct contains, instead the
T-Ag, the NcoI/CfoI fragment of the E6/E7 gene of the human
papilloma virus 16. The method to construct the E6/E7-plasmid is
based on Durst et al. (Durst et al. 1987, Oncogene 1:251-256).
After immortalization, the cells are passaged as necessary during
culturing and the resultant immortalized cells are then transferred
to a proliferation medium in step (vii). In the case of
keratinocytes, this transferral is preferably made at passage
2.
[0043] In step (viii) the immortalized cells are expanded in a
proliferation medium for immortalized keratinocytes or melanocytes
which will comprise a serum-free medium and preferably will
comprise NR-2 or NR-3 and M2-medium for melanocytes (described
infra). The immortalized cells are again cultured on continuously
precoated culture plates, with the coating again comprising a
solution of fibronectin, BSA and type 1 collagen.
[0044] After the immortalized cells are expanded in the
proliferation medium, they are then transferred in step (viii) to a
medium which provides for differentiation of normal and
immortalized keratinocytes. Preferably, this medium will comprise
NR-2 or modified MCDB 153 media containing high calcium, preferably
about 1.5 mM calcium, with culturing again being effected on
culture plates continuously coated with a solution of fibronectin,
BSA and type I collagen.
[0045] As noted above, it has been surprisingly discovered that
immortalized keratinocyte and melanocyte cell lines produced
according to the subject method maintain the ability to
differentiate and to express differentiation proteins which are
characteristic of normal differentiated keratinocytes and
melanocytes, even after high passage in tissue culture i.e. after
at least 10 passages. For example, the subject immortalized
keratinocytes express keratins as well as other proteins, e.g.
involucrin, filaggrin, and loricrin after high passage which are
either not expressed or are poorly expressed by previously reported
SV40 immortalized keratinocytes.
[0046] More specifically, several immortalized keratinocytes cell
lines produced according to the invention, DK2-NR, DK3-NR and
FK2-NR (see Tables 7 and 8 infra) express the differentiation
proteins keratin K1/10, keratin K14, involucrin, filaggrin and
loricrin even after in high passages (>passage 30).
[0047] Also, immortalized keratinocytes produced according to the
invention have a CYP450 profile which is similar if not identical
to that of normal human keratinocytes. For example, the subject
immortalized keratinocytes express CYP450 1A1, 2C, 2E1 and 3A5 but
do not express CYP450 1A2, 2A6, 2B6 and 2D6 which is characteristic
of the cytochrome 450 profile of normal keratinocytes. This was the
first time it could be demonstrated that normal and immortalized
human keratinocytes express CYP450 3A5 and not CYP450 3A4.
[0048] Also, the subject immortalized keratinocyte cell lines
express phase-II enzymes, e.g. glutathione-S-transferases (GST)
comparably to normal differentiated keratinocytes. More
specifically, the subject immortalized keratinocyte cell lines
express GST.alpha., GST.mu. and GST.pi. comparably to normal
keratinocytes.
[0049] Further, the subject immortalized keratinocytes express
enzymes and other proteins which are involved in cellular oxidation
and inflammatory responses comparably to normal keratinocytes. For
example, immortalized keratinocytes produced according to the
invention express superoxide dismutase (SOD). Also, in response to
phorbol esters immortalized keratinocytes produced according to the
invention express type I collagenase (a mediator of inflammation)
and TNF-.alpha.(tumor necrosis factor alpha).
[0050] The subject melanocytes have the capacity to express melanin
associated proteins and vimentin.
[0051] Moreover, the subject immortalized cell lines when grown in
organotypic culture form a highly stratified and polarized
epithelium having cornified superficial layers (stratum corneum)
even in higher passages (>passage 20). This has only been
previously reported for immortalized keratinocyte cell lines
established under conventional culture conditions, i.e. medium
containing serum, and using a feeder layer.
[0052] The subject immortalized cells lines are obtained under
total serum-free conditions without the use of any feeder layers
during culturing.
[0053] Moreover, as described in greater detail infra, it has been
surprisingly discovered that epinephrine is a strong growth factor
of normal keratinocytes when used in serum free medium.
Specifically, the NR-3 medium described infra contains epinephrine
which has been found to enhance the growth of normal keratinocytes
(see FIG. 1). This is quite surprising given the fact that
epinephrine has previously been reported to inhibit the growth of
keratinocytes (Halprin, J. Invest. Dermatol., 81:553-557 (1983)) or
to only have a moderate effect on keratinocyte cell growth (Koizumi
et al, J. Invest. Dermatol., 96:234-237, 1991).
[0054] Also, it has been surprisingly discovered that the
continuous coating of the culturing dishes or plates used to
culture primary and immortalized keratinocytes and/or melanocytes,
in particular with a coating or "cocktail" containing fibronectin,
BSA and type I collagen improves both the attachment of
keratinocytes and melanocytes to culture plates or culture dishes,
as well as enhancing cell growth. The use of such a coating
material has not been previously described for use with
immortalized keratinocytes and/or melanocytes.
[0055] As discussed, the present invention further specifically
provides a novel serum-free medium referred to as the NR-3 medium.
This medium allows for culturing and isolation of normal
keratinocytes and/or melanocytes from human skin under serum-free
conditions without the use of a feeder layer. This medium has been
found to improve the growth of normal keratinocytes and to allow
the establishment of normal keratinocyte cultures without any
contact with serum or feeder cells.
[0056] The exact composition of the NR-3 medium is described in
Table 1. This medium contains various amino acids, inorganic salts
as trace elements, vitamins, growth factors and other substituents.
For example, this medium contains as growth factors epidermal
growth factor (EGF recombinant), insulin, hydrocortisone,
transferrin (human), bovine pituitary extract, and epinephrine. As
noted, epinephrine has surprisingly been discovered to enhance the
growth of primary keratinocytes in tissue culture.
[0057] As amino acids this medium contains L-alanine,
L-arginine-HCI, L-asparagine-H.sub.2O, L-aspartic acid,
L-cysteine-HCl-H.sub.2O, L-glutamic acid, glutamine, glycine,
L-histidine-HCl-H.sub.2O, L-isoleucine, L-leucine, L-lysine-HCl,
L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,
L-tryptophan, L-tyrosine and L-valine.
[0058] The inorganic salts contained therein are ammonium
metavanadate, ammonium molybdate, calcium chloride, cupric sulfate,
ferric sulfate, magnesium chloride, manganese chloride, nickel
sulfate, potassium chloride, sodium acetate, sodium bicarbonate,
sodium chloride, sodium phosphate dibasic, sodium pyruvate, sodium
selenite, sodium silicate, tin chloride and zinc sulfate.
[0059] The vitamins contained in the NR-3 medium are d-biotin,
d-calcium pantothenate, choline chloride, cyancocalbumin, folic
acid, i-inositol, nicotinamide, pyridoxine and riboflavin.
[0060] The medium further contains adenine, ethanolamine,
phosphoethanolamine, phenol red Na, putrescine 2HCl, thiamine HCl,
thioctic acid, thymidine, glucose, HEPES and antibiotics
(fungizone, penicillin and streptomycin).
[0061] The preferred composition of the NR-3 medium is described in
Table 12. However, it is expected that the concentration of the
substituents contained in the NR-3 medium may be varied within wide
limits. More particularly, it is expected that the amounts of the
various substituents may be varied .+-.50 to .+-.0.1%, more
preferably from .+-.10 to .+-.0.1% from the concentrations
disclosed in Table 12. Moreover, it is expected that one or more of
the recited substituents may be deleted and other substituents may
be added provided that such substituents do not substantially
adversely affect the isolation and establishment of keratinocyte or
melanocyte primary cell cultures and immortalized cell lines. This
can be determined by one skilled in the art by trial and error
analysis.
[0062] As noted, a significant substituent of the subject NR-3
serum-free medium is epinephrine. It has been discovered that
epinephrine has a very strong growth promoting activity on primary
human keratinocytes.
[0063] The reason that epinephrine enhances proliferation of
keratinocytes is unclear. It has been reported that human
keratinocytes express enzymes for epinephrine synthesis and also
express at high density beta 2-adrenoreceptors (Schallreuther et
al., "Production of catecholamines in the human epidermis,"
Biochem. & Biophys. Res. Commun., 189:72-78 (1992)). These
enzymes are involved in the catecholamine biosynthetic pathway, in
particular phenylethanolamine-N-methyltransferase and biopterin
dependent tyrosine hydroxylase. By contrast, such enzymatic
activity cannot be detected in melanocytes and fibroblasts.
Accordingly, this enzymatic activity and/or receptor expression may
explain the ability of epinephrine to modulate the proliferation of
keratinocytes.
[0064] It is hypothesized by the present inventors that the NR-3
medium enhances the isolation and establishment of primary cell
cultures and cell lines because it suppresses cell differentiation
which leads to an enrichment of cells which maintain their ability
to differentiate and to express proteins and enzymes expressed by
normal differentiated keratinocytes and melanocytes.
[0065] More specifically, it is believed that the growth of
keratinocytes or melanocytes in serum containing medium favors
differentiation in the first passage. However, this is
disadvantageous (during the initial culturing period) because
differentiated cells do not grow well. This in turn results in the
overgrowth and selection of proliferative skin cells which only
possess a weak capacity to differentiate. Consequently, the number
of cells which possess high differentiation capacity are reduced if
serum is added to the medium prior to immortalization.
[0066] By contrast, in the present invention the keratinocytes and
melanocytes are cultured in a serum-free medium and under
conditions which inhibit the differentiation of melanocytes and
keratinocytes. In the present invention, serum-free medium is
preferably used during the entire cultivation period both prior to
and during immortalization, as well as during proliferation and
differentiation.
[0067] As noted, the subject NR-3 serum-free culture medium
inhibits differentiation of keratinocytes and thereby allows for
improved isolation of keratinocyte primary cell cultures and
immortalized cell lines derived therefrom. Moreover, this
serum-free medium contains low calcium concentrations which
inhibits selectively inhibits the growth of co-isolated
fibroblasts. This results in a highly selective growth medium which
favors the production of cultures which predominantly contain
melanocytes and keratinocytes. Accordingly, the subject serum-free
NR-3 medium is advantageous because it inhibits keratinocyte
differentiation and also inhibits fibroblast growth.
[0068] As discussed, a cell suspension produced from a single skin
sample which contains dissociated melanocytes, keratinocytes and
fibroblasts will preferably be cultured in the subject NR-3 medium.
This will be effected by seeding such cells onto culture dishes
which are continuously coated with a composition which facilitates
their attachment. Preferably, this coating will comprise a mixture
of fibronectin, bovine serum albumin and type 1 collagen. This
coating or "cocktail" coating has been previously described for
bronchial cells (Lechner et al., J. Tiss. Cult. Meth., 9:43-48
(1985)). The present inventors have found that this cocktail also
enhances the attachment of keratinocytes and melanocytes to plastic
culture dishes. Moreover, it has been surprisingly found that said
continuous coating of culture plates which are used to culture
primary and immortalized keratinocytes further improves cell
growth. The continuous coating of culture plates has not been
previously described for immortalized keratinocytes or
melanocytes.
[0069] During culturing the primary cell cultures are preferably
split when they reach or substantially reach confluence, and then
expanded onto other coated culture dishes. Typically, cell cultures
will be divided about every 10 to 14 days.
[0070] After the primary melanocytes and/or keratinocytes have been
cultured and expanded to desired cell numbers in NR-3 serum-free
medium using the described coated culture dishes they are then
immortalized. Preferably, the melanocytes and keratinocytes which
exhibit the best growth are immortalized. However, alternatively
the expanded primary melanocytes or keratinocytes may be used prior
to immortalization, e.g., in assays, skin grafting or in gene
therapy.
[0071] Immortalization of both melanocytes and keratinocytes may be
effected with a vector which provides for the expression of the
SV40 T-antigen or for the expression of the E6/E7 gene of human
papilloma virus 16 (HPV16). Preferably, immortalization is effected
by infection of melanocytes or keratinocytes with a retroviral
construct which provides for the expression of the SV40 T-antigen
or E6/E7 gene of HPV16. The cellular T-Ag infection is based
following the protocol of Pfeifer et al. Meth. Cell Sci. 17: 83-89,
1995 (except virus was collected from packaging cell line forwing
in DMEM, 10% fetal calf serum). During infection, serum containing
medium may be used. However, serum free medium is preferred for
melanocytes and keratinocytes, preferably, e.g., the PC-1 medium
described in the paper of Pfeifer et al., Meth. Cell. Sci., 14,
83-89 (1995), which reference is incorporated by reference herein.
Most preferably, immortalization is effected using the retroviral
construct referred to as pLXSHD+SV40(#328) shown in FIG. 2a, and
based on Pfeifer et al. and Stockshlaeder et al. (GeneBank, n.sup.o
accession M64753), or as pLXSHD+E6/E7 shown in FIG. 2b and based on
Durst et al. 1987, Oncogene 1, 251-256.
[0072] After immortalization the immortalized cell lines are
transferred to a proliferation medium, preferably NR-2 or NR-3, or
M2 (for melanocytes) using precoated culture dishes. After the
cells are proliferated to desired cell numbers, the cells are
transferred to a differentiation medium suitable for culturing
normal and immortalized keratinocytes. Preferably, this will
comprise NR-2 or modified MCDB 153 with high calcium (1.5 mM) or M2
(for melanocytes) using precoated cultured plates (the same BSA,
type I collagen, fibronectin coating).
[0073] As noted previously differentiated immortalized keratinocyte
and melanocyte cell lines produced according to the invention
exhibit improved properties which render these cell lines well
suited for use in assays which require differentiated human skin
cells. In particular, these cell lines have been found to express
proteins characteristic of normal differentiated melanocytes and
keratinocytes even after high passages.
[0074] For example, when immortalized keratinocytes produced
according to the invention are assayed by Western blot and RT-PCR,
they possess a cytochrome p450 (CYP450) profile similar if not
identical to normal keratinocytes. More specifically, immortalized
keratinocytes produced according to the invention express CYP450
1A1, 2C, 2E1, 3A5 and do not express CYP450 1A2, 2A6, 2B6 and 2D6.
This CYP450 profile is consistent with normal keratinocytes. Such a
metabolism profile has not previously been described for
immortalized keratinocytes. Indeed, this is the first time it could
be demonstrated that normal and immortalized keratinocytes express
CYP450 3A5 and not CYP450 3A4. Also, immortalized keratinocytes
produced according to the invention, when analyzed using antibodies
specific for differentiation markers, are found to express other
differentiation proteins even after high passage. More
particularly, the subject cell lines express the differentiation
proteins K1/10, keratin K14, involucrin, filaggrin and loricrin
even at high passages, i.e. after 10 passages and substantially
greater.
[0075] The subject, immortalized keratinocytes and melanocytes also
uptake exogenous essential fatty acids (EFA) and exhibit
desaturation and chain elongation of EFA highly consistent to
normal melanocytes and keratinocytes.
[0076] Further, as described in greater detail infra, the subject
immortalized keratinocytes express TNF(X and the inflammation
mediator collagenase type I, when treated with phorbol esters
comparably to normal keratinocytes. Also, the subject immortalized
keratinocytes express superoxide dismutase, an enzyme involved in
cellular oxidation similar to normal differentiated
keratinocytes.
[0077] Also, immortalized melanocytes produced according to the
invention treated with melanogenesis inducers (e.g., theophylline
and tyrosine) and melanogenesis inhibitors (kojic acid) respond
similarly to normal melanocytes.
[0078] Given these properties, the subject immortalized
keratinocytes and melanocytes are well suited for immunological,
pharmacological, photo- and chemotoxicological skin reaction
studies.
[0079] For example, the subject immortalized keratinocyte and
melanocyte cell lines and primary melanocytes and keratinocytes may
be used in assays which require differentiated skin cells, e.g.,
barrier-function studies (cornification) of reconstructed skin,
metabolism studies of differentiated keratinocytes (fatty acid
metabolism, antioxidant metabolism), studies relating to effects of
ultraviolet radiation on skin cells, studies relating to the
effects of potential skin irritants and sensitizers on skin cells,
assays measuring the effects of compounds on melanin production,
lipid-metabolism studies, topical treatment with xenobiotics (e.g.,
cosmetic oils, screening for putative protective compounds, e.g.,
photoprotectors), skin inflammation and irritation studies,
etc.
[0080] Also, immortalized keratinocyte and melanocyte cell lines
and primary melanocytes and keratinocytes produced according to the
invention are useful for screening potential anti-cancer treatment
compounds and skin disease treatment compounds. This will typically
involve exposing the cell line or primary cells to such compounds
for a period of time and ascertaining whether they induce any
adverse effects, e.g., genotoxicity, DNA adduct formation,
mutagenicity, cell transformation or cytotoxicity.
[0081] Also, the subject melanocyte and keratinocyte cell lines are
suitable for the expression of recombinant proteins, e.g., human
proteins and polypeptides, as well as for the production of RNA and
DNA.
[0082] Further, the subject immortalized cell lines have potential
utility for ex vivo genetic therapy. The subject cell lines should
provide a useful tool for genetic targeting and developing
genetically engineered cells which express desired gene products,
e.g., for therapeutic application, or for cell
toxicity/mutagenicity studies. Moreover, given the fact that the
subject cell lines closely mimic normal skin cells they should be
well suited for biosensitivity assays.
[0083] Additionally, primary keratinocytes and melanocytes produced
according to the subject invention, given that they are produced
under serum-free conditions, may be useful in gene therapy.
Essentially, because these cells are not exposed to serum, e.g.,
bovine or other animal serum (except for during viral infection and
storage in liquid nitrogen), they should be less subject to
potential contamination by viruses or other pathogenic agents.
Therefore, this should minimize the risk of these cells
transmitting pathogenic or infective factors during gene therapy.
Such ex vivo genetic therapy has potential in the treatment of
disorders such as Epidermolysis bullosa (keratin mutation
disorder), Vitiligo (a disorder involving melanin synthesis genes),
carcinomas and melanomas, allergic disorders and inflammation
related disorders. With respect to therapeutic treatment, the only
potential source of contamination is the bovine pituitary extract,
bovine insulin, bovine collagen, bovine serum albumin or human
fibronectin and human transferrin.
[0084] Also, the subject immortalized melanocyte and keratinocyte
cell lines and primary melanocytes and keratinocytes have utility
in DNA mutagenesis assays, skin mutagen screening assays, assays
for identifying chromosome damaging agents, malignant
transformation studies, cellular biochemistry studies (e.g., CYP450
activation assays), screening of compounds and compositions, e.g.,
essential fatty acid cocktails which are involved in inflammation
and allergic reactions, collagenase activation assays (related to
inflammation), involving TNF.alpha., interleukin detection.
[0085] A significant potential application of primary keratinocytes
or melanocytes produced according to the invention, given their
availability and method of production, is for skin grafting.
Because these primary keratinocytes and melanocytes are produced
under serum-free conditions, they should pose minimal risk of being
contaminated by pathogens (e.g. viruses) and infectious agents.
Moreover, because the subject melanocytes and keratinocytes may be
derived from an autologous host, i.e., a patient having a large
wound, this should minimize or eliminate the risk of rejection of
the skin graft, or other adverse immunological reaction, as well as
minimizing the risk of infection.
[0086] Examples of specific immortalized keratinocyte cell lines
produced according to the invention are FK2-NR, DK2-NR and DK3-NR
which were deposited on Oct. 5, 1995, at DSM-Deutsche Sammlung von
Mikrorganismen Und ZelIKulturen GmbH, which address is Mascheroder
Weg 1b D-38124 Branschweig Germany, and which have respectively
been accorded Accession Nos. DSM ACC2240, DSM ACC2238, and DSM
ACC2239. Furthermore examples of specific immortalized human
melanocyte lines produced according to the invention is DM2-NR
which is deposited on Dec. 11, 1996 at the Pasteur Institut, which
address is 25 rue de Docteur Roux 75724 Paris France, and which has
been accorded Accession No CNCM I-1796. These deposits were made in
accordance with the Budapest Treaty. All restrictions as to the
availability of these cell lines will be irrevocably withdrawn upon
issuance of a patent to this application or another application
which claims benefit of priority to this application.
[0087] Other features of the invention will become apparent in the
course of the following descriptions of exemplary embodiments which
are given for illustration of the invention and are not intended to
be limitative.
EXAMPLE 1
Characterization of Established Skin Cells
[0088] Table 1 lists all skin samples which were processed for
viral infection. The isolated keratinocytes which show the best
cell growth were used for immortalization.
1TABLE 1 Skin Samples Used for Cell Isolation in NR-3-Medium Origin
Name of Cell of Tissue Cell Strain Age Sex Growth.sup.1 thigh OS1
36 f ++ thigh OS2 68 f - thigh OS3 51 f + eyelid EL1 46 f + eyelid
EL2 49 f + abdomen Thor1 58 f - foreskin FK1-NR 5 m +++ foreskin
FK0-NR 13 m ++++ abdomen GK0-NR 26 f +++ breast DK0-NR 29 f +++
.sup.1Method: Cells were harvested in trypsin/EDTA (0.05%/0.01%)
and counted by using a hemocytometer, the results are the mean of
triplicates.
[0089] Human fibroblasts were isolated from the skin samples
FKO-NR, GKO-NR, DKO-NR. After the separation of the dermal and
epidermal compartment the dermis was cut into small pieces
0.2.times.0.2 mm and fixed on a 6 cm culture plate with serum.
Dulbecco's minimal essential medium (DMEM, 10% FCS) was added after
2-4 hours. This explant culture was then incubated until fibroblast
outgrowth was visible. Confluent fibroblast cultures were split and
expanded for frozen stocks.
EXAMPLE 2
[0090] 1) Characterization of Keratinocytes Growth in Serum-free
Media: primary cell cultures were cultivated in modified MCDB 153
[Boyce et al., J. Tissue Cult. Meth., 9:83-93 (1985); and Pittlekow
et al., J. Invest. Dermatol., 86:410-417, 1986] and NR-3 media. The
best cell growth has been observed in NR-3 medium (FIG. 1).
Improved cell growth has been also observed in fully defined NR-3
medium (NR-3 without bovine pituitary extract, BPE) compared to
modified MCDB 153 without BPE.
[0091] FIG. 1 comprises cell growth in NR-3 and
epinephrine-supplemented modified MCDB 153 (keratinocyte growth
medium) after 6 days. The modified MCDB 153 medium refers to
modified MCDB 153. Keratinocytes were harvested in trypsin/EDTA
(0.05%/0.01%) and counted by using a hemocytometer. The results
shown in FIG. 1 are the mean of triplicates.
[0092] 2) Effect of Coating on Cellular Attachment and Cell Growth:
the coating of culture plates was found to improve the cell
attachment and the cell growth of normal keratinocytes. In
particular, the results shown in Table 2 compare the growth of
keratinocytes in coated and uncoated culture plates. 100,000
keratinocytes were seeded on 3.5 cm plates containing NR-3
medium.
2TABLE 2 Effect of Surface Coating on the Cell Attachment attached
cells 24 h after seeding.sup.1 cell number after 4 d.sup.2 uncoated
% coated % uncoated coated DK0-NR 21.4 68.2 44600 143800 after
isolation DK0-NR 73.8 86.8 175500 282300 passage 2 .sup.1Number of
cells attached divided by the number of cells inoculated. Attached
cells were harvested in trypsin/EDTA (0.05%/0.01%) and counted by
using a hemocytometer. .sup.2Cells were harvested in trypsin/EDTA
(0.05%/0.01%) and counted by using a hemocytometer, the results are
the mean of triplicates.
EXAMPLE 3
[0093] 1) Immortalization of Keratinocytes: a cell suspension
produced from skin samples described in example 1, which contains
dissociated melanocytes, keratinocytes and fibroblasts, are
cultured in the subject NR-3 medium. This is effected by seeding
such cells onto culture dishes which are continuously coated with
the "cocktail" coating previously described for bronchial cells
(Lechner et al., J. Tiss. Cult. Meth., 9:43-48 (1985)). During
culturing the primary cell cultures when they reach or
substantially reach confluence, the cells are treated for 4 min
with trypsin/EDTA (0.025%/0.01%). During this treatment, the
melanocytes detached from the keratinocytes culture, and they are
collected separatly. Primary melanocytes and keratinocytes are thus
separated at this stage. After the primary keratinocytes have been
cultured and expanded to desired cell numbers in NR-3 serum-free
medium using the described coated culture dishes (promotes the
growth of keratinocytes versus melanocytes) they are then
immortalized. Immortalization of keratinocytes is effected with the
retroviral construct pLXSHD+SV40(#328) which provides for the
expression of the SV40 T-antigen (see Pfeifer et al. Meth. Cell
Sci. 17: 83-89, 1995; except virus was collected from packaging
cell line forwing in DMEM, 10% fetal calf serum). During infection,
the PC-1 serum free medium described in the paper of Pfeifer et
al., Meth. Cell. Sci., 14, 83-89 (1995), is used. After
immortalization the immortalized cell lines are transferred to the
NR-2 or NR-3 proliferation medium using precoated culture dishes.
After the cells are proliferated to desired cell numbers, the cells
are transferred to a differentiation medium suitable for culturing
normal and immortalized keratinocytes.
[0094] 2) Cell Proliferation of Immortalized Keratinocytes in High
Passages: the immortalized keratinocytes were demonstrated to
exhibit improved cell growth in high passages. This is shown in
Table 3 below. This was demonstrated by the estimation of the
population doubling time (PDT: time for one doubling of the cell
population during the logarithmic growth phase). Method:
Keratinocytes were harvested in trypsin/EDTA (0.05%/0.01%) and
counted by using a hemocytometer, the results are the mean of
triplicates.
3TABLE 3 Population Doubling Time (PDT) of Keratinocyte Lines Grown
in NR-3 Keratinocytes passage number PDT (h) crisis* in passage
FK2-NR 15 48.00 16-18 FK2-NR 39 21.16 DK1-NR 12 23.20 25-30 DK1-NR
15 31.05 DK1-NR 31 32.99 DK2-NR 15 22.26 20-21 DK3-NR 40 24.34 --
*Crisis: cell growth with reduced proliferation rate.
[0095] 3) CYP450-expression in Immortalized Human Keratinocyte
lines: CYP4501A1, 1A2, 3A5, 2E1, 2B6, 2A6, and 2D6 expression were
analyzed in normal and immortalized keratinocytes skin cells by
Western blot (protein-expression) and RT-PCR (mRNA-expression, see
Table 4). The expressed CYP450 in immortalized keratinocytes is
similar to normal keratinocytes. The rate of expression is slightly
reduced. However the DK2-NR-line shows almost a normal rate of
CYP450-expression. Method: RT-PCR (reverse transcriptase-polymerase
chain reaction) with specific primer for CYP450 (Mac et al. in
preparation).
4TABLE 4 CYP mRNA Expression in Human Keratinocytes Passage 1A2,
2A6, Keratinocytes number 1A1* 2C** 2E1 3A5 2B6, 2D6 FK0-NR 4 + + +
+ - FK2-NR 36 + + + + - DK0-NR 3 + + + + - DK1-NR 31 + + + + -
DK2-NR 13 + + + + - *1A1 mRNA expression in immortalized cell lines
was increased after induction with Benz(a)pyrene (1.5 .mu.M;
Amersham Inc.). The increase was comparable to normal cells
**2C17/19 and 2C18
[0096] 4) Response to CYP450-Inducers: the cell lines respond to
the CYP450-inducer Benz(a)pyrene like non-immortalized cells even
in high passages (see Table 5). This induction is not described for
T-Ag immortalized keratinocytes.
5TABLE 5 7-ethoxyresorufin O-deethylase (EROD; Sigma Inc.) Activity
In Human Keratinocytes after Induction with Benzpyrene (B(a)P)*
passage EROD Keratinocytes number (pmol/mg protein) FK0-NR 2
undetectable FK0-NR + B(a)P 2 0.58 FK2-NR 37 0.01 FK2-NR + B(a)P 37
1.05 DK0-NR 1 0.02 DK0-NR + B(a)P 1 1.03 DK1-NR 32 0.04 DK1-NR +
B(a)P 32 1.78 DK2-NR 14 0.02 DK2-NR + B(a)P 14 0.69 DK3-NR 39 0.01
DK3-NR + B(a)P 39 1.86 *Keratinocytes were incubated 24 h with
B(a)P (1.5 .mu.M). EROD activity was measured after incubation at
37.degree. C. by fluorescence detection of resorufin product (560
nm excitation 586 nm emission).
[0097] 5) Cell Differentiation: differentiation markers were
analyzed using specific antibodies. The specific antibodies used
are identified in Table 6. The highest differentiation capacity
could be demonstrated in DK2-NR and DK1-NR clone (Tables 7, 8).
6TABLE 6 Antibodies used for the Detection of Keratinocyte-Specific
Proteins Name of Specificity the Antibody Company/Reference T-Ag
Ab-2 Oncogene, Manhassat, NY Involucrin BTI BT-576 bti, Stoughton,
MA Filaggrin Filaggrin Paesel + Lorei, Frankfurt, Germany Loricrin
aAg 73 Magnaldo et al. 1992 Vimentin V9 Dako, Glostrup, Denmark
Keratin K4 6B10 Sigma, St. Louis, USA Keratin K7 LDS-68 Sigma, St.
Louis, USA Keratin K8 M20 Sigma, St. Louis, USA Keratin K10/1 K8.60
Sigma, St. Louis, USA Keratin K13 KS-1A3 Sigma, St. Louis, USA
Keratin K14 CKB1 Sigma, St. Louis, USA Keratin K17 CK-E3 Sigma, St.
Louis, USA Keratin K18 CY-90 Sigma, St. Louis, USA Keratin K19
A53-B/A2 Sigma, St. Louis, USA
[0098]
7TABLE 7 Detection of T-Ag and Differentiation Products of
Epidermal Keratinocytes Keratin- Passage ocyte Number T-Ag
Involucrin Filaggrin Loricrin Vimentin FK0-NR 2 - +++ ++ + ++
FK2-NR 25 +++ ++ ++ + ++ DK0-NR 2 - +++ +++ ++ +++ DK1-NR 13 +++
+++ ++ ++ ++ DK1-NR 30 +++ ++ ++ + ++ DK2-NR 11 +++ +++ +++ ++ +++
DK3-NR 36 +++ ++ ++ + ++
[0099]
8TABLE 8 Detection of Keratins (K) in Keratinocytes passage
Keratinocytes number K4 K7 K8 K10/1 K13 K14 K17 K18 K19 FK0-NR 2 ++
- ++ ++ +++ ++ ++ + + FK2-NR 25 +++ - ++ ++ +++ ++ ++ + + DK0-NR 2
++ - - +++ +++ +++ +++ - + DK1-NR 13 ++ - ++ +++ +++ ++ ++ + +
DK1-NR 30 + - ++ ++ ++ ++ + ++ + DK2-NR 11 +++ - +++ +++ +++ +++
+++ +++ ++ DK3-NR 36 + - +++ +++ +++ +++ +++ +++ ++ Method (Tables
7 and 8): Keratinocytes grown on chamber slides were stained after
fixation in pure methanol with antibodies listed in Table 6. +++:
highest concentration of protein, quantified by fluorescence
microscope. -: no protein expression.
[0100] 6) Expression of Glutathione-S-Transferase: the phase
II-enzyme Glutathione-S-transferase (GST) has been analyzed by
Northern blot and Western blot technique. All keratinocyte lines
express strongly mRNA for GST.pi. but not GST.alpha. and GST.mu.
(Table 9: Method: Northern Blot). The protein-expression profile of
GST.alpha., GST.mu. and GST.pi. in the cell lines was similar to
normal keratinocytes.
9TABLE 9 Protein-Expression of GST enzyme Keratinocytes GST.alpha.
GST.mu. GST.pi. FK2-NR - - +++ DK0-NR - - +++ DK1-NR - - +++ DK2-NR
- - +++ DK3-NR - - +++
[0101] 7) Essential Fatty Acid (EFA) Metabolism: to analyze and to
compare the desaturation and elongation of added EFA in
keratinocytes, immortalized (DK1-NR, FK2-NR) and normal
keratinocytes were treated with linoleic acid (LA, 15 .mu.M) and
.alpha.-inolenic acid (LN, 15 .mu.M). For these experiments the
EFA-deficient NR-2 (Biofluids Inc.) was used. Cell cultures were
treated after reaching confluency and shifting NR-2 high calcium
(1.5 mM). Cells were treated 4 days with EFA (renewed after 2
days).
[0102] EFA analysis was performed by extraction and separation of
phospholipids by TLC (thin layer chromatography) and quantification
of fatty acid-methylesters by GLC (gas liquid chromatography). The
formation of the desaturation and elongation products of LA
(20:4n-6 and 22:4n-6) and LN (20:5n-3, 22:5n-3 and 22:6n-3) could
be demonstrated. This metabolic profile was consistent with that
observed in normal keratinocytes.
[0103] 8) Karyotping: all cell lines were hypodiploid with most
chromosome counts in the dipoid range (except DK2-NR with
chromosome counts also in the hypotetraploid range). Cells other
than those of the analyzed cell lines were not detected in the
cultures. This result confirms the purity of the cell lines and the
absence of cellular contamination from other sources.
[0104] 9) In vivo Characterization: the tumorigenicity of
immortalized keratinocytes were determined by subcutaneous
injection (1-2mio keratinocytes) into nude mice. The tested
keratinocyte lines DK2-NR, DK3-NR, FK2-NR are not tumorigenic in
nude mice (4 months of incubation). DK3-NR was however weakly
tumorogenic in 6 animals among 10 animals after 5 months of
incubation.
[0105] 10) Response to Skin Irritants: the induction of the
"stress-gene" TNF.alpha. (tumor necrosis factor alpha) after
treatment with skin irritants PMA
(phorbol-12-myristate-13-acetate), SDS (sodium dodecyl sulfate),
DMSO (dimethyl sulfoxide), IL-1 .beta. (interleukin 1 beta), and
UV-B (ultraviolet B) was analyzed by Northern blot and biological
assays (Table 10). The keratinocyte lines respond to PMA and UV-B
and express the protein TNF.alpha. even in high passages.
[0106] After treatment of immortalized keratinocytes with phorbol
esters (PMA) an increase of collagenase (type I) expression was
observed.
10TABLE 10 Secretion of TNF.alpha. in Keratinocytes after Induction
with Skin Irritants Protein activity assay: .sup.3H-thymidine
incorporation into a TNF.alpha.-sensitive cells*. passage
TNF.alpha.-secretion after induction with Keratinocytes number PMA
SDS DMSO IL-1.beta. UV-B DK0-NR 3 + - - nt + DK1-NR 20-22 + + - + +
DK1-NR 32 + - - nt + DK2-NR 16 + - - - + DK2-NR 31 + - - nt + nt:
not tested *: sensitive cells: mouse fibrosarcoma cell line WEHI
164 clone 1.14 (ATCC).
[0107] 11) Organotypic Cultures: the cultivation of human
keratinocytes under organotypic conditions (keratinocyte grown air
exposed on collagen gel with feeder cells) was also conducted. All
keratinocyte lines showed a hyperproliferative morphology compared
with normal keratinocytes. These studies have been done in culture
medium with serum. The hyperproliferative cell growth was reduced
under serum-free conditions (NR-2 with high calcium (1.5 mM) on
plastic insert culture dishes without collagen gel and without
feeder cells).
EXAMPLE 4
[0108] 1) Immortalization of melanocytes: a cell suspension
produced from skin sample DKO-NR described in example 1, which
contains dissociated melanocytes, keratinocytes and fibroblasts,
are cultured in the subject NR-3 medium. This is effected by
seeding such cells onto culture dishes which are continuously
coated with the "cocktail" coating previously described for
bronchial cells (Lechner et al., J. Tiss. Cult. Meth., 9:43-48
(1985)). During culturing the primary cell cultures when they reach
or substantially reach confluence, the cells are treated for 4 min
with trypsin/EDTA (0.025%/0.01%). During this treatment, the
melanocytes detached from the keratinocytes culture, and they are
collected separatly. Primary melanocytes and keratinocytes are thus
separated at this stage. The collected primary melanocytes are then
seeded in NR-4 serum-free medium which specifically inhibits the
growth of keratinicytes. After the primary melanocytes have been
cultured and expanded to desired cell numbers in NR-4 serum-free
medium using the described coated culture they are then
immortalized. Immortalization of keratinocytes is effected with the
retroviral construct pLXSHD+SV40(#328) which provides for the
expression of the SV40 T-antigen (see Pfeifer et al. Meth. Cell
Sci. 17: 83-89, 1995; except virus was collected from packaging
cell line forwing in DMEM, 10% fetal calf serum). During infection,
the PC-1 serum free medium described in the paper of Pfeifer et
al., Meth. Cell. Sci., 14, 83-89 (1995), is used. After
immortalization the immortalized cell lines are transferred to the
M2 proliferation and differentiation medium (DMEM!F 12 medium,
Biofluids, No148; can also be bought from M. Olssen, Uppsala,
Sweden).
[0109] 2) Characterization of T-Ag Expressing Human Melanocytes:
the expression of melanin-associated proteins of immortalized
melanocytes produced according to the invention (especially DM2-NR)
was compared to normal melanocytes. It was demonstrated that
immortalized cells expressed melanin-associated proteins, melanoma
associated antigen (MAA) and HMB45 similar to normal cells,
although at lesser levels of expression.
[0110] 3) Induction of Melanin Synthesis: the melanogenesis of T-Ag
expressing melanocyte cell lines (especially line DM2-NR) was
compared with normal melanocytes. The melanocytes were treated with
the melanogensis inducers tyrosin and theophylline and the
melanogenesis inhibitor kojic acid. The melanocyte lines
(especially DM2-NR) responded to melanogenesis modulator comparable
to normal cells. The induction/inhibition of melanogenesis was also
demonstrated to be dose dependent.
EXAMPLE 5
[0111] the strain DKO-NR described in example 1 has been
immortalized, as described above, with the HPV16 based retrovirus
construct pLXSHD+E6/E7 which is depicted in FIG. 2b. Several
continuous keratinocytes lines were selected. The analysis results
of these lines in terms of differentiation products (cytokeratins,
GST, TNF.alpha., Involucrin, Filaggrin, Loricrin, Vimentin) are
similar to those obtained for the lines DK2-NR and DK3-NR.
EXAMPLE 6
[0112] The composition of the novel NR-3 medium of the invention
and several other serum-free medium of the present invention, i.e.
NR-1, NR-2 and NR-4, are compared below.
[0113] 1) Media Composition of NR-1 Medium (see table below)
11 NR1 [milligram/liter] Amino Acids L-Alanine 9.0000 L-arginine,
HCl 316.0000 Asparagine, H.sub.2O 15.0000 L-Aspartic acid 4.0000
L-Cysteine, HCl, H.sub.2O 42.0000 L-Glutamic acid 14.8000 Glutamine
877.0000 Glycine 7.6000 L-Histidine, HCl, H.sub.2O 50.4000
L-Isoleucine 98.4000 L-Leucine 131.2000 L-Lysine, HCl 36.6000
L-Methionine 13.4000 L-Phenylalanine 14.9000 L-Proline 34.6000
L-Serine 126.2000 L-Threonine 23.8000 L-Tryptophan 9.2000
L-Tyrosine 13.6000 L-Valine 70.2000 Inorganic Salts Ammonium
Metavanadate [NH.sub.4VO.sub.3] 0.0006 Ammonium Molybdate
[(NH.sub.4).sub.6Mo.sub.7O.sub.24 .times. 4H.sub.2O] 0.0010 Calcium
Chloride [CaCl.sub.2 .times. 2H.sub.2O] 16.2000 Cupric Sulfate
[CuSO.sub.4 .times. 5H.sub.2O] 0.0025 Ferric Sulfate [FeSO.sub.4
.times. 7H.sub.2O] 1.4000 Magnesium Chloride [MgCl.sub.2 .times.
6H.sub.2O] 122.0000 Manganese Chloride [MnCl.sub.2 .times.
4H.sub.2O] 0.0002 Nickel Sulfate [NiSO.sub.4 .times. 6H.sub.2O]
0.0003 Potassium Chloride [KCl] 112.0000 Sodium Acetate 301.5000
Sodium Bicarbonate [NaHCO.sub.3] 1088.0000 Sodium Chloride [NaCl]
5200.0000 Sodium Phosphate Dibasic [Na.sub.2HPO.sub.4 .times.
7H.sub.2O] 536.0000 Sodium Pyruvate 55.5000 Sodium Selenite
[Na.sub.2SeO.sub.3] 0.0050 Sodium Silicate [Na.sub.2SiO.sub.3
.times. 9H.sub.2O] 0.1420 Tin Chloride [SnCl.sub.2 .times.
2H.sub.2O] 0.0001 Zinc Sulfate [ZnSO.sub.4 .times. 7H.sub.2O]
0.5100 Vitamins d-Biotin 0.0200 d-Calcium Pantothenate 0.2600
Choline Chloride 28.0000 Cyanocobalamin (B12) 0.4100 Folic Acid
0.7900 i-Inositol 18.0000 Nicotinamide (B3) 0.0400 Pyridoxine (B6
.times. H.sub.2O) 0.0600 Riboflavin (B2) 0.0400 Others Adenine
27.3000 Epidermal growth factor (EGF, human recomb.) 0.0010
Ethanolamine 0.0310 Glucose 1080.0000 HEPES 6000.0000
Hydrocortisone 0.5000 Insulin (bovine) 5.0000 Phenol Red 1.2000
Phosphoethanolamine 0.0710 Putrescine 2HCl 0.1600 Thiamine HCl
0.3400 Thioctic Acid 0.2100 Thymidine 0.7300 Transferrin (human)
10.0000 Osmolarity 280-285 mOsm/kg
[0114] 2) Media Composition of NR-2 Medium: same as NR-1 but
supplemented with bovine pituitary extract (Biofluid Inc.) at 35
mg/l, and containing antibiotics (Gibco BRL, Life Technologies
Inc.) fungizone (0.25 mg/l) penicillin (10.000 units/l) and
streptomycin (10 mg/l).
[0115] 3) Media Composition of NR-3: same substituents as NR-2 but
supplemented with epinephrine (Biofluid Inc.) 250 .mu.g/l.
[0116] 4) Media Composition of NR-4: same substituents as NR-2 but
supplemented with .beta.FGF (3 .mu.g/l) (basic fibroblast growth
factor obtained from Sigma Inc.) and phorbol
12-myristate-13-acetate (10 .mu.g/l) (PMA) (C.C.R. Inc.).
[0117] Although the invention has been described in considerable
detail with regard to certain preferred embodiments thereof, other
embodiments within the scope of the teachings of the present
invention are possible. Accordingly, neither the disclosure nor the
claims which follow, are intended, nor should be construed to be,
limited by the description of the preferred embodiments contained
herein.
* * * * *