U.S. patent application number 12/717096 was filed with the patent office on 2012-03-22 for methods for producing nonadherent avian cell lines.
This patent application is currently assigned to VIVALIS. Invention is credited to Fabienne Guehenneux, Bertrand PAIN.
Application Number | 20120070893 12/717096 |
Document ID | / |
Family ID | 27763651 |
Filed Date | 2012-03-22 |
United States Patent
Application |
20120070893 |
Kind Code |
A9 |
PAIN; Bertrand ; et
al. |
March 22, 2012 |
METHODS FOR PRODUCING NONADHERENT AVIAN CELL LINES
Abstract
The present invention relates to a method for producing avian
cell lines, comprising gradual or complete withdrawal of growth
factors, serum and/or feeder layer so that the established lines
are adherent or nonadherent cells capable of proliferating
indefinitely in a basic culture medium. The invention also relates
to the cells derived from such lines which are particularly useful
for the production of substances of interest.
Inventors: |
PAIN; Bertrand; (Lyon,
FR) ; Guehenneux; Fabienne; (Orvault, FR) |
Assignee: |
VIVALIS
Roussay
FR
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20100221825 A1 |
September 2, 2010 |
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Family ID: |
27763651 |
Appl. No.: |
12/717096 |
Filed: |
March 3, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12350781 |
Jan 8, 2009 |
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12717096 |
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10625847 |
Jul 24, 2003 |
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12350781 |
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PCT/FR03/00735 |
Mar 7, 2003 |
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10625847 |
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Current U.S.
Class: |
435/349 |
Current CPC
Class: |
C12N 2500/92 20130101;
C12N 2510/02 20130101; C12N 5/0606 20130101; C12N 2500/90 20130101;
C12N 2500/95 20130101 |
Class at
Publication: |
435/349 |
International
Class: |
C12N 5/071 20100101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2002 |
FR |
0202945 |
Claims
1-53. (canceled)
54. A method for producing a nonadherent avian cell line capable of
proliferating in a basal medium in the absence of exogenous trophic
factors and cytokines, said method comprising culturing embryonic
stem cells obtained from blastodermal disks of fertilized eggs in a
culture medium, wherein: (a) in at least the first passage, said
embryonic stem cells are cultured in a primary medium comprising:
(i) the trophic factor insulin-like growth factor 1 (IGF-1) and the
cytokine ciliary neurotrophic factor (CNTF); (ii) an inactivated
feeder comprising mouse fibroblast STO cells; and (iii) a basal
medium, wherein the basal medium is supplemented with fetal calf
serum at an initial concentration of from 8 to 12%; (b) the cells
obtained in step (a) are further cultured in a primary medium that
has been modified by progressively depriving the primary medium of
said trophic factors and said cytokines defined in (i) of step (a),
wherein the progressive withdrawal of each trophic factor and
cytokine is carried out by successive passage of the cells in a
culture medium having a lower concentration of at least one of said
trophic factors or said cytokines as compared to the culture medium
of the prior passage, until the medium is free of all exogenous
trophic factors and cytokines; and (c) the cells obtained in step
(b) are inoculated at a high density into a bacteriological dish to
produce a nonadherent avian cell line capable of proliferating in a
basal medium in the absence of exogenous trophic factors and
cytokines.
55. The method of claim 54, wherein the nonadherent avian cell line
obtained in step(c) is capable of proliferating in a basal medium
in the absence of exogenous trophic factors and cytokines for at
least 50 days.
56. The method of claim 54, further comprising the following step
(d): (d) proliferating in suspension the nonadherent cells derived
from the lines obtained in step (c) in a medium free of exogenous
trophic factors and cytokines
57. The method of claim 54, wherein the cells derived from the
lines obtained in step (c) have a high nucleocytoplasmic ratio, an
endogenous alkaline phosphatase activity, an endogenous telomerase
activity, and a reactivity with specific antibodies selected from
the group of antibodies SSEA-1, (TEC01) and EMA-1.
58. The method of claim 54, wherein the cells used in step (a) are
obtained by suspending cells from blastodermal disks of fertilized
eggs in a culture medium comprising the tropic factors and
cytokines as defined in (i) of step (a), wherein said cells are
inoculated into a feeder comprising mouse fibroblast STO cells,
incubated, and then collected.
59. The method of claim 54, further comprising the following step
(d): (d) proliferating cells derived from the lines obtained in
step (c) in a basic medium selected from the group consisting of
DMEM, GMEM, HamF12 and McCoy, wherein the medium is supplemented
with various additives selected from the group consisting of
nonessential amino acids, vitamins and sodium pyruvate.
60. The method of claim 54, wherein the primary medium of step (a)
further comprises any one or more of the following trophic factors
and cytokines: stem cell factor (SCF), basic fibroblast growth
factor (bFGF), interleukin 6 (IL-6), soluble IL-6 receptor (sIL-6r)
and interleukin 11 (IL-11).
61. The method of claim 60, wherein the primary medium of step (a)
further comprises the following trophic factors and cytokines: stem
cell factor (SCF), basic fibroblast growth factor (bFGF),
interleukin 6 (IL-6), and soluble IL-6 receptor (sIL-6r).
62. The method of claim 60, wherein the primary medium of step (a)
further comprises the following trophic factors and cytokines: stem
cell factor (SCF) and interleukin 11 (IL-11).
63. The method of claim 60, wherein the primary medium of step (a)
further comprises the following trophic factors and cytokines: stem
cell factor (SCF), basic fibroblast growth factor (bFGF),
interleukin 6 (IL-6), soluble IL-6 receptor (sIL-6r) and
interleukin 11 (IL-11).
64. The method of claim 54, wherein said embryonic stem cells
obtained from blastodermal disks of fertilized eggs are chicken
embryonic stem cells.
65. The method of claim 54, wherein the basal medium in step a)
(iii) is a basal medium selected from the group consisting of DMEM,
GMEM, HamF12 and MacCoy medium.
Description
[0001] The present invention relates to a method for producing
avian cell lines, in particular avian stem cells, comprising
progressive or total withdrawal of growth factors; serum and/or
feeder layer. These spontaneously established lines are adherent or
nonadherent cells capable of proliferating indefinitely in a basic
culture medium. The invention also relates to the cells derived
from such lines which are particularly useful for the production of
vaccines and of substances of interest.
[0002] Stem cells are cells identified by their culture in vitro
from an embryo, from part of an embryo or even from an adult
tissue. The expression stem cell is understood to mean any
pluripotent cell of embryonic or adult origin which has a capacity
for self-renewal and is capable of giving specialized
differentiated cells. In other words, any noncancerous cell capable
of dividing indefinitely in culture and of giving a daughter cell
having the same capacity for proliferation and differentiation as
the mother cell from which it is derived. These isolated cells
exhibit particular morphological and immunocytochemical
characteristics. It is also possible to distinguish the notion of:
[0003] embryonic stem cells (CES cells), stem cells which have the
characteristic feature of being obtained from culturing parts or
all of a very early embryo (blastula stage). These CES cells
exhibit in vitro all the characteristics of a stem cell, and in
vivo the unique capacity of contributing to the morphogenesis of an
embryo and of participating in germline colonization when they are
reimplanted in any manner whatsoever in a recipient embryo. [0004]
somatic stem cells (SSC), cells which have all the characteristics
of stem cells when they are cultured in vitro, but which, unlike
CES cells, do not have the potential to colonize in vivo the gonads
after infection into an embryo. They contribute solely to the
morphogenesis of the somatic tissues in the embryo.
[0005] Unlike already differentiated primary cells, stem cells do
not exhibit an easily identifiable characteristic state of
morphological differentiation (fibroblasts, adipocytes, macrophage,
and the like), but are rather characterized by a state of
proliferation and of nondifferentiation. This state results
different behaviors such as a rapid proliferation in vitro, a
characteristic morphology, the presence of different markers,
variable requirements for growth factors and an ability to respond
to particular stimuli for induction of differentiation. They are
not sensitive to replicative senescence, a critical period for a
large number of differentiated primary cells, including the
fibroblasts for example.
[0006] To maintain avian stem cells in vitro for long periods of
time, it is necessary to observe specific culture and maintenance
conditions as described in Pain et al., 1996; U.S. Pat. No.
6,114,168 and EP 787 180.
[0007] The in vitro culture of a primary cell under satisfactory
medium and growth factor conditions allows it to proliferate only
for a certain number of passages. This cell can be obtained
directly from a dissociated tissue or from part of this tissue.
This number of passages is nevertheless highly dependent on the
animal species considered, on the origin of the tissue, on the age
of the donor organism and the like.
[0008] In most cases, the cell proliferation observed in vitro
slows progressively and then the cells stop proliferating.
[0009] This arrest often corresponds to a replicative senescence,
known by the term Hayflick limit. This stoppage is thought to be
the result of the action of a true molecular clock of which one of
the key components is thought to be the length of the telomeres.
The telomeres are repeat sequences situated at the end of the
chromosomes. The shortening of these repetitive nucleotide
structures is the consequence of the replication of DNA on a
semiconservative mode. In the absence of the telomerase enzyme,
which is in charge of adding the repeat sequences at the end of the
chromosomes, a point of no-return is reached with regard to the
size of the telomeres, a point beyond which an as yet unknown
molecular mechanism for activation of genes involved in controlling
the cell cycle is triggered. The cells are then thought to be
blocked in the G1 phase in their divisions and are thought to stop
proliferating. Numerous factors appear to be involved in this
negative control of the cell cycle such as various cyclins,
specific kinases, RB and P53 proteins, specific transcription
factors such as E2F and many others (mdm2, BTG, p21, and the like).
Conversely, the telomerase enzyme can therefore be viewed as a
central factor in cell immortality because it maintains the length
of the telomeres and therefore makes the cell insensitive to this
loss caused by successive divisions.
[0010] In an organism under development and during the life of this
organism, only a few cell types, including certain lymphocytes,
exhibit a permanent expression of telomerase. This activity also
appears to be one of the characteristics of stem cells, both at the
somatic level (SSC) and at the germline level. This property of
expressing, of maintaining of expression and of "awakening"
expression of the telomerase activity is also often associated with
the immortal character of a cell maintained in vitro. To date,
numerous cancer cells are also detected positive for telomerase
activity. This activity is thought to be partially responsible for
the capacity for uncontrolled proliferation of tumor cells in
vivo.
[0011] This telomerase activity is, in all cases, an excellent
marker for the stem cell character and for the germline lineage and
for the capacity of a cell to become immortal. Two criteria are
therefore used: the telomerase activity and the size of the
telomeres.
[0012] In an establishment perspective and very briefly, according
to the literature and the results already available in many
laboratories, the establishment of cell lines may be carried out
according to two routes: a spontaneous establishment resulting from
noninduced intrinsic genetic damage or a triggered establishment,
induced by the use of viruses, retroviruses or by other means such
as chemical agents, irradiation, UV (ultraviolet) radiation, and
the like. In mammals, for example, the establishment of rodent
(mouse, rat, and the like) cells is recognized as being fairly easy
spontaneously; on the other hand, the situation is quite different
for human cells regardless of their tissue origin (Smith and
Pereira-Smith, 1996).
[0013] Thus, when it is desired to obtain cell lines from the
abovementioned avian stem cells so as to produce a mass of
substances of interest in vitro, the problem is to be able to
maintain cells in culture in an economical medium while avoiding
stumbling blocks such as cellular differentiation and
senescence.
[0014] At the avian level, it is generally accepted that the
establishment of primary cells is a rare, or even practically
nonnexistent event. The only notable published exception appears to
be the DF-1 line which results from the immortalization, described
as being spontaneous, of chicken fibroblasts (Foster et al., 1991;
patent U.S. Pat. No. 5,672,485, ATCC No. CRL 12203). At the level
of this line, recent articles mention the first components of
observed deregulation (Kim et al., 2001a; Kim et al., 2001b).
[0015] In the immortalization process, a first step leads the
proliferating cell to the Hayflick limit which, depending on the
cell types, is between 10 and 50 passages. A first spontaneous
mutational event then takes place which allows the cell to cross
this first blockage, an event which often affects the p53 and pRb
genes, and the like. The cells therefore continue to proliferate
until the moment when a second blockage occurs, which is in general
lifted by new mutations in other genes and by the activation of
telomerase, which is often observed.
[0016] At the avian level, it is in general accepted that the
establishment of immortal primary cells is a practically
nonexistent event. Accordingly, a large number of lines have been
obtained by culturing tumor cells, often directly taken from a
biopsy of the tumor. This obtaining in vitro is in fact the result
of the impairment in vivo of certain genes, which are responsible
for the appearance of the tumor. An example is provided by the
fibroblast line SB-CEV-1, which is isolated from the culturing of a
visceral tumor from an animal (ATCC No. CRL 10497, U.S. Pat. No.
5,846,527). This approach is greatly facilitated by the existence
of a very large number of avian viruses and retroviruses which have
been identified, isolated and often characterized at the molecular
level. Being often oncogenic by their direct or indirect action
(activation of a transforming endogenous gene during their
integration, expression of the oncogenic protein(s) endogenous to
the virus), these viruses cause tumors and lesions; thus, the
obtaining of cell lines in different differentiated lineages is
possible from the culturing of the infected organs of the animals.
The in vitro use of these viruses and retroviruses, isolated in
vivo, has been developed in addition. There may thus be mentioned
nonexhaustively: [0017] the lymphoblastoid lines DT40 and. DT95,
obtained in the presence of the avian leukosis virus (ALV) and in
which the myc locus is activated (Baba et al., 1985, ATCC No. CRL
2111, CRL 2112), [0018] the turkey lymphoblastoid line MDTC-RP19,
established with the Marek's disease virus (U.S. Pat. No.
4,388,298, ATCC No. 8135), [0019] the lymphoblastoid line ConA-C1,
established with the REV virus (reticuloendothelial virus, ATCC No.
12135, U.S. Pat. No. 5,691,200), [0020] the myelomonocytic line BM2
established with the MH2 virus (Liu et al., 1977, U.S. Pat. No.
5,388,680), [0021] and a whole series of hematopoietic lines
obtained with different viruses, [0022] the erythroblastic line HD4
(6C2), obtained with the AEV virus [0023] the monocytic line HD11
(Beug et al., 1979), obtained with the MC29 virus [0024] the
granulocytic line HD13 (Golay et al., 1988), obtained with the E26
virus [0025] the mixed hematopoietic line IID57 (Metz and Graf,
1991) also obtained with the E26 virus.
[0026] However, the problem in these transformation approaches is
the obtaining of cells which produce viruses and carry the
activated genome of the viruses and retroviruses used. These
activations and this viral presence are brakes on their industrial
use as replication support for viruses of interest or for the
production of specific proteins under optimum safety
conditions.
[0027] Another approach to overexpression of oncogenes, of
immortalizing genes (adenovirus E1A gene, polyoma SV40 "large T",
and the like) or gene fragments has also made it possible to obtain
lines from already differentiated primary cells. These components
may be introduced into the cells by simple transfection of a vector
allowing the expression of the immortalizing part, but may also be
introduced via viruses or retroviruses which have been genetically
modified to express these immortalizing components. The origin of
the immortalizing components may be avian or otherwise, viral or
otherwise. The tropism for avian cells can in fact be linked to the
original virus or can also be modified. By way of example, the duck
fibroblast line TDF-2A is thus obtained by introducing a first
immortalizing gene and then an antiapoptotic gene (Guilhot et al.,
1993, patent U.S. Pat. No. 6,255,108). Other methods have been
developed, such as the overexpression of p53 (Foster et al., U.S.
Pat. No. 5,830,723).
[0028] In addition, the action of chemical carcinogens directly on
the animal according to different modes of administration has
allowed, inter alia, the obtaining [0029] of the QT6 and QT35
lines, of quail fibroblasts (Moscovici et al., 1977, ATCC No. CRL
1708), [0030] of the chicken hepatocyte line LMH (Kawaguchi et al.,
1989, ATTC No. CRL 2117), [0031] of the chicken fibroblast line
CHCC-OU2 (ATCC No. CRL 12302, U.S. Pat. No. 5,989,805).
[0032] The expression immortalization event is understood to mean
various actions such as: [0033] the action of oxidative, heat or
chemical stress capable of inducing modifications in the physiology
of the cells and/or mutations, [0034] the action of the products of
specific genes in the physiology of the cells, such as certain
immortalizing genes (oncogenes, protooncogenes, cell cycle genes,
antiapoptotic genes and the like), [0035] targeted destruction by
functional recombination or inactivation of antioncogenes,
apoptotic genes, tumor suppressor genes, antiproliferative genes,
leading to a functional deregulation of the cell cycle or of the
physiology of the cell, [0036] the control of proliferation genes
by their functional blocking, and the like, [0037] the action of
rays (UV, gamma, X, and the like), [0038] the action of chemical
mutagens (substances which damage DNA, substances similar to growth
factors, and the like), [0039] the conjugated action of these
various actions taken separately.
[0040] In the context of the invention, it has been found that the
withdrawal of growth factors, serum and/or feeder layer leads to
the isolation of populations of stem cells, in particular of
somatic stem cells, which can grow indefinitely in basic culture
media.
[0041] In addition, apart from the hematopoietic stem cells which
are for the most part nonadherent cells, the majority of the cells,
obtained according to the prior art techniques mentioned above
(fibroblasts, hepatocytes, and the like), exhibit an adherent
phenotype. Now, the industrial use of cells, as viral replication
support, favors nonadherent cells. This phenotype is advantageous
both because of ease of handling which avoids the use of a
proteolytic enzyme for dissociation and for the high densities
reached by nonadherent cells cultured in vitro.
[0042] The present invention describes the production of lines
which can become spontaneously nonadherent and for which the
nonadherence is obtained by a withdrawal of the feeder layer.
Because of their growth in suspension, these lines are perfectly
suitable for industrial use for the production of substances of
interest in bioreactors.
[0043] In addition to their properties of growing on a basic
culture medium, it has been discovered that these cell lines allow
the replication of certain viruses in yields equivalent to or even
higher than the yields obtained with current methods, which makes
these cells particularly useful for the mass production of
vaccines.
DESCRIPTION
[0044] Thus, in a first aspect, the present invention relates to a
method for producing avian cell lines, characterized in that it
comprises the following steps:
[0045] a) culturing avian cells in a medium containing all the
factors allowing their growth and an inactivated feeder layer,
[0046] b) passage by modifying the culture medium so as to obtain
progressive or total withdrawal of said factors, of the serum
and/or of the feeder layer,
[0047] c) establishing adherent or nonadherent cell lines capable
of proliferating in a basal medium in the absence of exogenous
growth factors, serum and/or inactivated feeder layer.
[0048] In the context of the invention, the expression
"establishment of a line" is understood to mean maintaining cells
in culture in vitro over a considerable period of time.
Advantageously, the cells derived from the lines obtained in step
c) are capable of proliferating for at least 50 days, 100 days, 150
days, 300 days or preferably at least 600 days. The 600 days do not
constitute a time limit because the cell lines obtained are still
alive after much longer time periods. Hence, these lines are
considered as being able to grow indefinitely in a basic culture
medium free of exogenous growth factors, serum and/or inactivated
feeder layer. The expression "line" is understood to mean any
population of cells capable of proliferating indefinitely in
culture in vitro while retaining to a greater or lesser degree the
same morphological and phenotypic characteristics.
[0049] The cells derived from the lines according to the invention
may be avian stem cells, in particular avian somatic stem
cells.
[0050] The stem cells according to the invention can serve to
obtain differentiated cell lines. Indeed, these stem cells have the
property of being pluripotent, that is to say that they have the
potential to be induced in multiple differentiation pathways which
can be characterized by various specific markers.
[0051] These cells can also be precursor cells, which correspond to
the partially differentiated cells of an adult or embryonic tissue,
by contrast to a stem cell and which is capable of dividing and of
giving more differentiated cells. The expression "differentiated
cell" is understood to mean any specialized cell of an adult or
embryonic tissue, having specific markers or fulfilling specific
physiological functions. It is possible, in a particular aspect of
the invention, in particular for particular isolates or clones
derived from a particular isolate obtained during establishment,
for these stem cells to contribute to the germline. In this case,
these stem cells established as lines are thought to be embryonic
stem cells.
[0052] Of course, the method mentioned above makes it possible to
obtain cellular clones derived from cells obtained from established
lines. These clones are cells which are genetically identical to
the cell from which they are derived by division.
[0053] In a particular embodiment, the invention relates to a
method as defined above, in which the established lines are
adherent stem cells which proliferate in the absence of inactivated
feeder layer.
[0054] In this regard, in the method described above, step b)
consists in a withdrawal of the components of the medium (growth
factors alone or serum alone or growth factors and then serum or
alternatively serum and then growth factors). We also found that it
is possible to obtain non adherent cells after several passages, at
any moment, from these adherent stem cells that proliferate with or
without feeder layer.
[0055] In another embodiment, the invention relates to a method as
defined above in which the established lines are nonadherent stem
cells which proliferate in suspension in a medium free of exogenous
growth factors.
[0056] In this regard, in the method described above, step b)
consists in a progressive or total withdrawal of the feeder layer
and then optionally in a withdrawal of the other components of the
medium (growth factors and serum).
[0057] In another embodiment, the invention relates to a method as
described above in which the established lines are nonadherent stem
cells which proliferate in suspension in a medium free of serum
(serum-free medium).
[0058] In another embodiment, the invention relates to a method as
defined above, in which the established lines are nonadherent stem
cells which proliferate in suspension in a medium free of exogenous
growth factors and serum.
[0059] In another alternative, step b) consists in a progressive or
total withdrawal of the growth factors, optionally followed by a
progressive withdrawal of the serum.
[0060] In another alternative, step b) consists in a progressive or
total withdrawal of the growth factors and/or serum, optionally
followed by a withdrawal of the feeder layer.
[0061] In addition, the established lines may be cells which
proliferate in a serum-depleted medium, in particular in a medium
free of serum. The expression serum-depleted is understood to mean
a gradual reduction of the concentration of serum spread out over
time. This method allows a selection of clones which adapt to these
new, increasingly drastic conditions until stable lines are
obtained which are capable of growing in a serum-depleted medium or
in a medium completely free of serum.
[0062] The method described above may additionally comprise a step
in which the cells obtained in step c) are subjected to a selection
in culture media used for large-scale production so as to obtain
clones suitable for the production of vaccines intended for human
or animal therapy.
[0063] The cells according to the invention have at least one of
the following characteristics: [0064] a high nucleocytoplasmic
ratio, [0065] an endogenous alkaline phosphatase activity, [0066]
an endogenous telomerase activity, [0067] a reactivity with
specific antibodies selected from the group of antibodies SSEA-1
(TEC01), SSEA-3, and EMA-1.
[0068] Preferably, the cells of the invention have all the
abovementioned characteristics.
[0069] In an additional aspect, the invention relates to a method
for producing avian lines, which is mentioned above, in which the
cells derived from the lines obtained in step c) are modified in
order to allow a better use in vitro such as the extension of the
greater life span or growth densities or alternatively of the lower
nutrient requirements.
[0070] Advantageously, the cells derived from established lines are
modified in order to produce a substance of interest, in particular
a polypeptide of interest, an antibody or an attenuated virus. Said
cells may be modified by any technique accessible to persons
skilled in the art, in particular homologous, directed and/or
conditional recombination (Cre-Lox or FLP-FRT system), by
transformation with any vector, plasmid, in particular with the aid
of retroviruses.
[0071] The medium used in step a) may comprise at least one factor
selected from cytokines, in particular LIF, IL-11, IL-6, IL-6R,
CNTF, Oncostatin and other factors such as SCF, IGF-1 and bFGF.
[0072] In addition, the inactivated feeder layer used in step a) is
preferably composed of fibroblasts, including mouse fibroblasts
established as a line. Among these fibroblasts are in particular
the STO cells which may or may not be modified or transfected with
expression vectors (Pain et al., 1996). In this method, the cells
used in step a) are cells obtained by suspending cells obtained
from blastodermal disks of fertilized eggs in a culture medium
comprising at least one cytokine, b-FGF, and SCF. Said cells are
inoculated into a layer of feeder cells, incubated, and then
collected.
[0073] Step b) consists in a progressive withdrawal of each growth
factor added to the medium in step a), in particular a cytokine,
b-FGF, and SCF, comprising a passage in a new medium free of at
least one of said factors and in repeating various successive
passages until the medium is free of all of said factors. The
expression progressive withdrawal is understood to mean a removal
factor by factor from the culture medium. Alternatively, it is
possible to carry out a drastic or total withdrawal, that is to say
the removal of all of said factors all at once. Thus, the
withdrawal of step b) may consist in progressively reducing the
concentration of one or more factors or in culturing the avian stem
cells directly in a medium free of one or more factors or
alternatively free of all of said factors.
[0074] Step b) may also comprise the withdrawal of the serum. In
this regard, the withdrawal may be progressive, by reducing the
serum concentration during each passage, for example on passing
from 10% to 7.5% and then 3.75% and 2%, tending toward 0%
(serum-free medium). Alternatively, a drastic withdrawal may be
carried out.
[0075] Step b) may also comprise the withdrawal of the feeder
layer. The withdrawal of the feeder layer may also be gradual, by
reducing the number of inactivated feeder cells during each
passage. Alternatively, it is possible to carry out a drastic
withdrawal.
[0076] Of course, the order of withdrawals can vary. For example,
it is possible to start with the withdrawal of the growth factors
and continue with the withdrawal of the feeder layer.
[0077] Thus, in another aspect, the invention relates to the
established cell lines and to the cells derived from said lines
which can be obtained from the method described above, said cells
being capable of proliferating for at least 50 days, 100 days, 150
days, 300 days, or preferably at least 600 days in a medium free of
exogenous growth factor, serum and/or feeder layer.
[0078] These cell lines and the cells derived therefrom are capable
of proliferating for at least 50. days, 100 days, 150 days, 300
days, or preferably at least 600 days in a basal medium, in
particular in a medium such as DMEM, GMEM, HamF12 or McCoy
supplemented with various additives commonly used by persons
skilled in the art. Among the additives, there may be mentioned
nonessential amino acids, vitamins and sodium pyruvate.
[0079] The invention also relates to the cell lines and the cells
derived from such lines described above, characterized in that they
are avian stem cells, in particular avian somatic stem cells or
avian embryonic stem cells.
[0080] These stem cells may be adherent, while proliferating in the
absence of the inactivated feeder layer. Alternatively, these stem
cells are nonadherent and proliferate in suspension in a basal
medium mentioned above.
[0081] These cells are also characterized in that they have at
least one of the following characteristics: [0082] a high
nucleocytoplasmic ratio, [0083] an endogenous alkaline phosphatase
activity, [0084] an endogenous telomerase activity, [0085] a
reactivity with specific antibodies selected from the group of
antibodies SSEA-1 (TEC01), SSEA-3, and EMA-1.
[0086] Advantageously, these cells are genetically modified so as
to produce a substance of interest, in particular a polypeptide of
interest, an antibody or an attenuated virus.
[0087] Cells of the invention can for example support the
replication of live or attenuated viruses, in particular the
viruses selected from the group of adenoviruses, hepadnaviruses,
herpesviruses, orthomyxoviruses, papovaviruses, paramyxoviruses,
picornaviruses, poxviruses, reoviruses and retroviruses.
[0088] Preferably, the viruses belong to the family of
orthomyxoviruses, in particular the influenza virus, to the family
of paramyxoviruses, in particular the measles, mumps and rubella
viruses.
[0089] In another embodiment, the viruses replicated on these cells
belong to the to the family of poxvirus, in particular canarypox
virus, fowlpox virus as well as vaccinia virus.
[0090] Thus, the invention relates to the cell lines described
above, the cells derived from said lines and also the cell lines
obtained from cells which have been genetically modified.
Preferably, the invention relates to the cell lines derived from
step c) of the method described above, characterized in that they
are avian stem cells capable of growing indefinitely in a basal
medium free of exogenous growth factors, depleted of serum or free
of serum and/or of feeder layer.
[0091] In another aspect of the invention, the cells obtained at
the end of step c) may be genetically modified.
[0092] The invention also relates to a cell culture comprising
cells derived from the cell lines described above, in particular
avian stem cells or avian embryonic stem cells, and a basal medium
free of exogenous growth factors depleted of serum or free of serum
and/or of inactivated feeder layer.
[0093] In an additional aspect, the invention relates to the use of
the cell lines and cells described above for the production of
substances of interest, in particular of proteins of therapeutic
interest, for the replication of live or attenuated viruses, in
particular viruses chosen from the group of adenoviruses,
hepadnaviruses, herpesviruses, orthomyxoviruses, papovaviruses,
paramyxoviruses, picornaviruses, poxviruses, reoviruses and
retroviruses.
[0094] Preferably, the cell lines and the cells described above are
used for the production of viruses belonging to the family of
orthomyxoviruses, in particular the influenza virus, and for the
production of viruses belonging to the family of paramyxoviruses,
in particular the measles, mumps and rubella viruses.
[0095] It is possible to introduce into these lines and cells, used
for supporting the replication of live or attenuated viruses, the
component(s) necessary for accomplishing the complete viral cycle
of the virus so as to obtain, for example, the overexpression of
the receptor for the virus at the surface of the cell.
[0096] Therefore, one best mode of the invention is to use the
cells as defined above to produce live or attenuated vaccine, for
example recombinant vaccine, comprises culturing the adherent or
non adherent cell lines established in step c) according to the
process described above, inoculating said cells with viral
particles and culturing said cells in a basal medium as mentioned
above until cell lysis occurs and newly produced viral particles
are released in said medium. The invention has shown to be
particularly useful for the production of attenuated virus
belonging to the family of poxvirus, in particular canarypoxvirus,
fowlpoxvirus and vaccinia virus such as such as native or
recombinant vaccinia virus (for example Modified Vaccinia virus
Ankara, MVA (such as MVA available under ATCC Number VR-1508) or
other orthopoxviruses) and is further described in the examples
below.
[0097] Furthermore, the invention is aimed at the use of the cells
according to the invention for producing recombinant viruses
expressing antigens as vaccine against infection diseases such as
smallpox and cancer (for example melanoma, prostate cancer, breast
cancer, lung cancer, ovary cancer, liver cancer . . . ).
[0098] For the remainder of the description, reference will be made
to the legend to the figures below.
LEGEND
[0099] FIGS. 1-3: Growth curves for the cell lines of the invention
(with withdrawal of serum (FIG. 2) and with withdrawal of feeder
layer (FIG. 3).
[0100] FIGS. 4: Photograph, showing the characteristic morphology
of avian stem cells
[0101] N: nucleus, n: nucleolus and C: cytoplasm
[0102] (isolate S86N99, X40 magnification, photograph taken with a
Sony Cyber-shot digital camera)
[0103] FIG. 5: Photograph showing the alkaline phosphatase activity
of avian stem cell lines which are adherent or which are in
suspension
[0104] After fixing (0.1% formaldehyde/0.5% glutaraldehyde, 30
minutes at 4.degree. C.), the cells are rinsed twice in 1.times.PBS
and incubated for between 10 and 30 minutes at 37.degree. C. in an
NBT/BCIP (Nitro Blue Tetrazolium chloride 0.375 mg/ml,
5-bromo-4-chloro-3-indolyl phosphate 0.188 mg/ml, 0.1M Tris pH 9.5,
0.05M MgCl.sup.2, 0.1M NaCl) solution. The reaction is stopped by
two 1.times.PBS washes and the photographs are taken.
[0105] A--illustrates the characteristic violet coloration of the
endogenous alkaline phosphatase activity obtained with the adherent
line S86N45 p87, a line cultured with no feeder or factor
(.times.40 magnification, Sony Cyber-shot digital camera).
[0106] B--illustrates the violet coloration characteristic of the
endogenous alkaline phosphatase activity obtained with the EB14
line maintained from 8 passages in suspension, line derived from
the S86N45 cells, cultured in suspension with no feeder or factor
(.times.20 magnification, Sony Cyber-shot digital camera).
EXAMPLE 1
Variable Origin of the Live Material Used
[0107] The establishment of cell lines is often greatly linked to
the genetic nature of the cell material. Thus, in the murine
example, proportionately few genetic bases are permissive to the
production of embryonic stem ES cells and often involves a notion
of inbred animals. In the case of avian animals, it is difficult to
obtain inbred animals for historical reasons and because of the
origin of selection of commercial strains; it being of interest
precisely to avoid inbreeding. Eggs are the initial source of the
cells cultured in this invention. The eggs are preferably used
nonincubated, but a few hours of incubation may be necessary in
order to obtain the first stages of development of the embryo. The
cells obtained are derived from different chicken strains. Among
the strains used, there may be mentioned the S86N strain, a
commercial strain intended for the production of chicken bearing a
quality label, CNRs, the strain intended for the production of
chicken bearing a quality label, Marens, a local strain which is
genetically and phenotypically well characterized, White Leghorns,
a strain more intended for the production of eggs for consumption
and a reference strain for research laboratories, and the like. In
the latter strain, various origins have been tested including
certain eggs (called Valo) obtained from the White Leghorn strain
from Lohmann (Germany) considered to be "SPF" (Specific Pathogen
Free) eggs kept under very particular health safety conditions.
Numerous cell isolates were obtained from various strains,
suggesting the general character of the method.
EXAMPLE 2
Production and Establishment of the Adherent Cells
[0108] The eggs are opened, the yolk is separated from the egg
white during the opening. The embryos are removed from the yolk
either directly or with the aid of a Pasteur pipette, or with the
aid of a small absorbent filter paper (Whatmann 3M paper), cut out
beforehand in the form of a perforated ring with the aid of a
punch. The diameter of the perforation is about 5 mm. These small
rings are sterilized using dry heat for about 30 minutes in an
oven. This small paper ring is deposited on the surface of the yolk
and centered on the embryo which is thus surrounded by the paper
ring. The latter is then cut out with the aid of small pairs of
scissors and the whole removed is placed in a Petri dish, filled
with PBS or with a physiological saline. The embryo thus carried
away by the ring is cleaned of the excess yolk in the medium and
the embryonic disk, thus freed of the excess vitellin, is collected
with a Pasteur pipette.
[0109] In both cases, the embryos are placed in a tube containing
physiological medium (1.times.PBS, Tris Glucose, medium, and the
like). The embryos are then mechanically dissociated and inoculated
on a "feeder" into defined culture medium. Among the preferred
conditions used for the culturing, preference is given to the
culture medium composed of MacCoy medium as basal medium
supplemented with fetal calf serum at an initial concentration of
12 to 8%, with nonessential amino acids at 1%, with a mixture of
vitamins of commercial origin at 1%, with sodium pyruvate at a
final concentration of 1 mM, with beta-mercaptoethanol at a final
concentration of 0.2 mM, glutamine at a final concentration of 2.9
mM, with an initial mixture of antibiotics containing gentamycin at
a final concentration of 10 ng/ml, penicillin at a final
concentration of 100 U/ml and streptomycin at a final concentration
of 100 .mu.g/ml. Rapidly after the first passages of the cells, the
mixture of antibiotics is no longer added to the medium. The
expression rapidly is understood to mean after the first 3 to 5
passages in general. A mixture of nucleosides may also be added,
this mixture being prepared as described above (Pain et al., 1996).
Among the basal media tested under these same conditions and which
give similar results are the HamF12, Glasgow MEM and DMEM media,
the latter supplemented with biotin at a final concentration of 8
mg/l. By way of comparison, the biotin concentration is 0.2 mg/1 in
the MacCoy medium, 0.0073 mg/1 in the HamF12 and 0 in the
commercial DMEM and GMEM media.
[0110] The growth factors and the cytokines added to the culture
medium are preferably factors and cytokines which are recombinant,
including mouse SCF at a final concentration of 1 ng/ml, IGF-1 at a
final concentration of 1 to 5 ng/ml, CNTF at a final concentration
of 1 ng/ml, IL-6 at a final concentration of 1 ng/ml, and the
soluble IL-6 receptor at a final concentration of 0.5 ng/ml to 1
ng/ml. In some experiments, some other factors may be added during
the first passages. For example up to passage 3 or 10, it is
possible to add bFGF to the medium at a final concentration of 1
ng/ml and IL-11 at a final concentration of 1 ng/ml.
[0111] The inoculation is carried out into this medium on the
inactivated "feeder" composed of mouse fibroblasts established as
lines, the STO cells. In some cases, these cells were transfected
with simple expression vectors allowing the expression of growth
factors such as avian SCF, constitutively in the STO cells. Thus,
this "feeder" produces the factor in a form which is soluble and/or
attached in the plasma membrane of the cells.
[0112] After initial inoculation of the cells directly into this
medium, the medium is partially changed the next day, and then
partially or completely during subsequent days, depending on the
rate of adhesion observed for the primary cells. After about 4 to 7
days depending on the cases, the initial culture is dissociated and
transferred into new dishes in the same initial medium on the
inactivated feeder. After three to five passages, the cells are
cultured on an inactivated feeder of STO cells which are
nontransfected or transfected with an expression vector encoding a
resistance to an antibiotic such as the gene for resistance to
neomycin, to hygromycin, to puromycin and the like. After about
twenty passages, the cells are progressively deprived of growth
factors and cytokines. The expression gradual withdrawal is
understood to mean a removal factor by factor from the culture
medium. Thus, at one passage, SCF is first of all removed, and
then, two or three passages later, IGF-1. If the cells do not
exhibit morphological alterations or a variation in their average
rate of proliferation, the other factors, such as CNTF and IL-6,
are then removed. This withdrawal may also be drastic. All the
factors are in this case removed all at once. The cells are then
observed and are only passaged several days later if their rate of
proliferation is modified. The latter solution is generally that
which is practiced.
[0113] Various isolates are thus obtained and maintained for very
long periods of time. The expression very long periods of time is
understood to mean periods of the order of several weeks with a
minimum of 50 days, preferably periods greater than. 200 to 400
days, without limitation in time. Periods greater than 600 days are
observed.
[0114] Regardless of the support used, all the cells which are
adherent are dissociated with a proteolytic dissociation enzyme,
such as pronase, collagenase, dispase, trypsin, and the like.
Preferably, a proteolytic enzyme of bacterial origin is used in
order to avoid any potential contaminant of animal origin.
[0115] These cells have the characteristics of embryonic stem cells
with a specific morphology illustrated, by way of example, by the
photograph of FIG. 4 i.e. a small size, a large nucleocytoplasmic
ratio, a nucleus with at least one nucleolus which is clearly
visible and a very small cytoplasm. These cells are characterized
by growth in the form of more or less compact solid masses. The
adherent and nonadherent cells exhibit cross-reactivity with a
number of antibodies, as described above in Pain et al., 1996 and
in patents U.S. Pat. No. 6,114,168 and EP 787 180. The endogenous
telomerase activity component is also present and is an important
factor in the "stem" nature of these cells.
[0116] Cells of different isolates are obtained and maintained for
long periods of time.
[0117] Table 1 illustrates a few of the characteristics of these
isolates
TABLE-US-00001 Name Species Start "Stoppage" Days Passage
Generation S86N16 Chicken S86N 26-01-2000 05-08-2001 559 207 692
WL3 Chicken WL 28-06-2000 09-08-2001 403 153 333 Valo4 Chicken Valo
26-09-2000 07-02-2002 401 135 317 S86N45 Chicken S86N 29-01-2001
12-11-2001 287 118 329
[0118] It will be noted that the term "stoppage" does not
correspond to the end of the proliferation of the cells but to a
deliberate stoppage of the cell cultures by the experimenter. The
number of generation n is obtained by the formula X=2.sup.n or X is
the theoretical cumulative number of cells. This number is
available since the cells are counted at each passage and during
each inoculation. The complete history of the culture is thus
available.
EXAMPLE 3
Passage of the Cells
[0119] One of the characteristics of stem cells, in particular
somatic stem cells and embryonic stem cells, is their capacity to
proliferate in vitro for considerable periods of time. In order to
propagate and to passage the cells, the culture medium is changed
and replaced with fresh medium a few hours before their passage.
The curve presented in FIG. 1 illustrates a profile of cell growth
and establishment.
EXAMPLE 4
Doubling Time and Average Division Time
[0120] Starting with the established cells in culture and the cells
presented in the preceding examples, a mean division time can be
calculated. For all the independent isolates obtained, the rate of
proliferation increases slightly during successive passages, thus
causing the average division time during the establishment of the
cells to vary. In the adherent phase, the cells are initially
inoculated on an inactivated feeder layer and are passaged
regularly at a constant initial inoculation density of 1 to
2.times.10.sup.6 cells per 100 mm dish. Table 2 illustrates the
doubling time (d) and the mean division time (MDT in hour) for 3
established cell types as a function of the culture time. It is
observed that the mean doubling time decreases during the
establishment.
TABLE-US-00002 TABLE 2 days Cells 50 100 150 200 250 300 350 400
450 500 550 S86N16 (d) 0.30 0.63 1.00 0.86 1.13 1.15 1.47 1.70 1.94
1.50 1.9 S86N16 (MDT) 80 38 24 27.9 21.2 20.9 16.3 14.1 12.4 16
12.6 S86N45 (d) 0.49 0.89 0.89 1.45 2.15 x x x x x x S86N45 (MDT)
49 26.8 27 16.5 11.1 x x x x x x Valo4 (d) 0.03 0.61 1.00 1.17 1.26
1.03* 1.08* 1.25* x x x Valo4 (MDT) >48 39.3 24 20.5 19 23.3
22.2 19.2 x x x The mean doubling time d is established for the
period of time indicated in days with the following formula: d =
(1/Log2 .times. (LogX2/X1)) .times. 1/(T2 - T1) where X2 and X1 are
total numbers of cells at the times T2 and T1. This formula is the
direct consequence of the calculation of the number of generations
N by the formula X = 2.sup.n presented in example 1. The mean
division time (MDT) is then obtained in hours by dividing 24 hours
by d. *The Valo cells are passaged during this establishment on a
plastic support without the presence of a feeder. The doubling time
decreases and then increases again, when the cells become
rehabituated to this new environment.
EXAMPLE 5
Control of the Level of Serum for the Proliferation of the
Lines
[0121] During the obtaining of these lines, the culture media used
are conventional culture media comprising a base (DMEM, GMEM,
HamF12, McCoy, and the like) supplemented with various additives
such as nonessential amino acids, vitamins, and sodium pyruvate.
This complex medium comprises fetal calf serum, which remains a
central component of the culture, even though components of
different origins, including plant components, can be gradually
used. A process for controlling and habituating the cells to
relatively low proportions of fetal calf serum is presented. It is
thus possible to maintain cells in high proliferation (division
time>1) with low percentages of serum (2% for example in the
case of the S86N16 cells).
[0122] The curves presented in FIG. 2 illustrates the relative
reduction of serum for a given cell type: S86N16 cells. The
doubling time and the mean division times were also calculated and
presented in table 3. It will be noted that the mean division time
increases as a function of the relative reduction in serum. A
recovery phase is nevertheless observed after some time in culture
under the conditions mentioned. This time remains nevertheless less
than 24 h (d>1), which already represents a very advantageous
proliferation in industrial terms even at serum concentrations of
2%, which is already relatively low. Improvements with regard to
the different metabolites to be used may be envisaged in order to
increase this time and still further optimize the culture
conditions.
TABLE-US-00003 TABLE 3 Condition 10% 7.5% 3.75% 2% d 2.02 1.51 1.47
1.08 MDT 11.9 15.8 16.3 22.2
[0123] The examples are taken between passages p204 and p179 for
the 10% condition, between p198 and p176 for the 7.5%, between p224
and p201 for the 3.75% and between p216 and p199 for the 2%.
EXAMPLE 6
Deprivation of the Cells of Feeder Layer
[0124] Under the initial culture conditions, the presence of a
layer of inactivated cells appears to be necessary in order to
obtain embryonic stem cells as was described above. This feeder
layer no longer appears to be necessary after a number of passages.
Only the "culture treated" plastic appears to be important. Indeed,
one of the characteristics of some eukaryotic cells is to
proliferate in adherent form. In order to facilitate the adhesion
of the cells, the various plastic materials used are "culture"
treated. They undergo during their manufacture a treatment which
adds charges at the surface of the plastic, which charges promote
the adhesions of the extracellular matrix of the cells. By
contrast, the cell culture untreated plastic, often called plastic
of bacteriological quality, is not surface treated by addition of
specific feeders. The adhesion of the cells thereto is generally
very difficult, or even impossible, or then induces changes in
morphology, and in behavior which are often drastic. This
distinction between the two plastic qualities makes it possible to
obtain, depending on the inoculations which are carried out
therein, cells with different behaviors. Gradual deprivation of the
cultures of inactivated "feeder" makes it possible to obtain, after
a few passages, homogeneous cultures of stem cells directly
inoculated on "culture treated" plastic.
[0125] The comparative growth curves for the cells maintained in
the presence and in the absence of inactivated "feeder" are
presented with the case of the S86N16 cells in FIG. 3. This
adaptation of the cells is progressive so as not to lose the stem
cell character of the cells initially maintained on a "feeder".
Progressive derivatives are thus made. The obtaining of cells which
proliferate on plastic is the accomplishment of the withdrawal
process. In table 4, the division times show sensitivity of the
cells to their environment. As in the case of the progressive
withdrawal of serum, an adaptation is obtained with a recovering
effect on the cells after a few passages under the conditions
defined.
TABLE-US-00004 TABLE 4 Condition 1.2 0.5 0.3 plastic d 1.95 1.84
1.39 1.42 MDT 12.3 13 17.3 16.9
[0126] The examples are taken between the passages p154 and p131
for the 3 conditions 1.2.times.10.sup.6, 0.5.times.10.sup.6 and
0.3.times.10.sup.6 feeder cells and between p161 and p139 for the
condition on plastic alone.
EXAMPLE 7
Deprivation of the Cells in Growth Factors
[0127] Under the initial culture conditions, the presence of growth
factors is necessary. It is possible to schematically distinguish
two families of factors: the cytokines and the trophic factors.
[0128] The cytokines are mainly cytokines whose action is through a
receptor which is associated with the gp130 protein. Thus, LIF,
interleukin 11, interleukin 6, CNTF, oncostatin and cardiotrophin
have a similar mode of action with the recruitment at the level of
the receptor of a specific chain and the combination of the latter
with the gp130 protein in monomeric or sometimes heterodimeric
form. In a few cases, the combination of a soluble form of the
receptors, a form described inter alia for the receptors for
interleukin 6 and CNTF, makes it possible to increase the
proliferative effect observed. It has been previously shown that
the addition of at least one of these cytokines appeared to be
necessary for obtaining embryonic stem cells.
[0129] The trophic factors are mainly SCF, IGF-1 and bFGF, which
are also used at the start of the Culture, as described above.
Their presence is also necessary for obtaining and amplifying the
cells.
[0130] By progressively reducing these growth factors, it is
possible to obtain, after a few passages, culture conditions which
allow the proliferation of the embryonic or somatic stem cells
without the addition of an exogenous growth factor. The different
markers used to characterize these cells are always positive for
the cells maintained with no factors.
EXAMPLE 8
Comparison of the Media Used
[0131] Inoculated into different media, the cells are not obtained
with the same frequencies. Comparison of the compositions of the
media makes the identification of one of the components in
particular difficult. It appears more likely that the whole
combination allows an improvement in the physiology of the cells.
Among the preferred media, the Ham F12 medium, the MacCoy medium,
the DMEM medium and a DMEM medium enriched with biotin will be
noted. Starting with such an isolate, adaptation trials are carried
out in these different media.
EXAMPLE 9
Establishment of the Nonadherent Cells
[0132] During the successive passages of the stem cells, a
high-density inoculation directly into the bacteriological dish
makes it possible to obtain, after a few passages, embryonic cells
which become detached from their substrate and which proliferate in
suspension in the form of small regular aggregates. This
proliferation is encouraged over several passages by mere dilution,
mechanical dissociation and nonuse of proteolytic enzyme. The
stirring of the cultures is generally carried out but does not
represent a distinguishing factor for obtaining nonadherent cells.
Like the adherent cells, these cells have a characteristic
morphology of stem cells, i.e. a small size, a large
nucleocytoplasmic ratio, a nucleus with at least one nucleolus
which is clearly visible and a very small cytoplasm. These cells
are characterized by a growth in small aggregates which are more or
less compact. These nonadherent cells exhibit cross-reactivity with
a number of antibodies, as described above in Pain et al., 1996.
These cells are also positive for the endogenous telomerase
activity (as presented in example 10 for the EB1, EB4 and EB5
cells). In a nonadherent phase, the cells exhibit a high
proliferation in different media. The initial inoculation density
and the very regular supply of fresh medium provides high densities
which may range above 1.times.10.sup.6 cells per ml. Table 5
summarizes the main characteristics of a few isolates (parental
cells, initial passage of the making into a suspension, number of
days maintained in culture in suspension, number of passages and of
generations obtained before voluntary stoppage of the
maintenances). It can thus be noted that the passage for the making
into a suspension can vary from one isolate to another (see isolate
EB1 and EB14) and the proliferation rate (see isolate EB3 and
EB14).
TABLE-US-00005 TABLE 5 Parental Initial Name cells passage Start
Days Passages Generations EB1 S86N16 p111 20-01-2001 184 41 120 EB3
S86N16 p118 23-01-2001 381 17 40 EB4 S86N45 p100 25-09-2001 44 17
40 EB5 S86N45 p100 25-09-2001 44 17 40 EB14 S86N45 p81 05-09-2002
70 24 65
[0133] It will be noted that the term "start" corresponds to the
cells being placed under nonadherence.
EXAMPLE 10
Characterization of the Established Cells
[0134] The stem cells maintained for long culture times are
characterized with the same criteria as those described above (Pain
et al., 1996). It is thus possible to regularly detect the
endogenous alkaline phosphatase activity, illustrated by the
photograph of FIG. 5, the endogenous telomerase activity and
reactivity with specific antibodies such as the antibodies SSEA-1
(TEC-01) and EMA-1.
[0135] One of the important criteria during the establishment of
the cells is the presence of telomerase. Various tests were carried
out during the maintenance of the cells in culture using a TRAP
detection kit (Telomerase PCR Elisa, Roche). The cells are detected
positive after various passages in culture. Thus, the telomerase
activity is detectable for the S86N16 cells, the S86N45 cells and
for the EB1, EB4 and EB5 cells which are derived therefrom in a
nonadherent form (see table 6). The CEFs (Chicken Embryonic
Fibroblasts) maintained in primary culture are considered as
negative. The threshold of an OD<0.2 is the threshold
recommended by the kit as the negative threshold. All the analyses
were carried out on an equivalent of 2000 cells.
TABLE-US-00006 TABLE 6 Assay of the telomerase activity in various
lines at various passages Cells Passage Telomerase OD S86N16 p12
1.7 p29 2.8 p185 0.97 p204 0.95 S86N16 EB1 p134 1.1 S86N45 p50 0.87
p58 1.1 p66 0.96 p94 1.2 S86N45 EB4 p112 1.4 S86N45 EB5 p112 0.94
CEF* p4 0.07
EXAMPLE 11
Transfection and Induction of the Cells
[0136] The stem cells maintained in a growth over the long term are
transfected with various expression plasmids. It has been shown
that avian stem cells could be transfected (Pain et al., 1996). In
particular, the nonadherent cells are transfected and various
sorting systems make it possible to identify the stably transfected
cells (cell sorting, limiting dilution, and the like). These
genetic modifications can be made at the undifferentiated stage of
the stem cell. Once this modification has been obtained, the cell
is then induced to differentiate spontaneously or by addition of a
differentiation inducer. In this case, it is possible to use
retinoic acid at concentrations of 10.sup.-8 M to 10.sup.-6 M, or
dimethyl sulfoxide at concentrations of 1. to 2% final or sodium
butyrate at concentrations of 10.sup.-4 to 10.sup.-8 M, or phorbol
ester (TPA, PMA, and the like) or lipopolysaccharides (LPS) at
concentrations of 1 to 5 .mu.g/ml final. In another example, the
cells can form embryoid bodies in suspension, which embryoid bodies
can be caused to adhere to plastic after dissociation or
nondissociation of the cells constituting them. These
differentiated cells then proliferate but have a more limited
capacity for proliferation over the long term. By targeting the
genetic modification on a gene which influences the proliferation
of the cells, it is possible to make these differentiated cells
capable of proliferating over the long term.
EXAMPLE 12
Infection of the Cells
[0137] The adherent and nonadherent cells can be infected with
different viruses and retroviruses including avian viruses and
retroviruses. These cells can thus serve as a replication support
for the production of viral stocks intended for the production of
live, attenuated or inactivated human and veterinary vaccines
depending on the cases. Among the viruses of interest, there may be
mentioned those of the family of adenoviruses (such as Human
Adenovirus C, Fowl Adenovirus A, Ovine Adenovirus D, Turkey
Adenovirus B), circoviridae (such as Chicken Anemia Virus, CAV),
certain coronaviruses, such as avian infectious bronchitis virus
(IBV), flaviviruses (such as Yellow fever virus and hepatitis C
virus), hepadnaviruses (such as Hepatitis B virus and
Avihepadnaviruses such as Duck hepatitis B virus); herpesviruses
(such as Gallid herpesvirus, HSV (Herpes simplex virus) and Human
herpesvirus 1, 3 and 5), orthomyxoviruses (such as the influenza
virus: Influenzavirus A, Influenzavirus B and Influenza-virus C),
papovaviruses (such as polyomavirus and more particularly Simian
virus 40), paramyxoviruses (such as measles, mumps and rubella
viruses and such as respiroviruses and pneumoviruses such as human
respiratory syncytial virus and Metapneumovirus such as Avian
pneumovirus), picornaviruses (such as polio virus, hepatitis A
virus, and such as Encephalomyocarditis virus and foot-and-mouth
disease virus), poxviruses (such as fowlpox virus and avipox
viruses including Canarypox viruses, Juncopox viruses, Mynahpox
viruses, Pigeonpox viruses, Psittacinepox viruses, Quailpox
viruses, Sparrowpox viruses, Starlingpox viruses, Turkeypox
viruses), orthopoxvirus such as vaccinia virus, MVA, and reoviruses
(such as rotaviruses), retroviruses (such as ALV, avian leukosis
virus, Gammaretroviruses such as Murine leukemia virus,
Lentiviruses such as Human immunodeficiency virus 1 and 2) and
Togaviridae such as Rubivirus, in particular Rubella virus.
EXAMPLE 13
Protocol for Infecting a Nonadherent Avian Cell Line (EB1) with a
Virus
[0138] Amplification of the Cells:
[0139] The EB1 or EB14 cells are inoculated into a medium,
preferably MacCoy's 5A, HAMF12 or DMEM medium, or any other medium
of interest, containing 5% serum at a concentration of
0.2.times.10.sup.6 cells/ml for an initial volume of 50 ml in
general. They are maintained in culture at 39.degree. C. and at
7.5% CO.sub.2, with stirring. Fresh medium is added every day for
the 3 to 4 days for which the amplification lasts in order to reach
a cell concentration of 1 to 3.times.10.sup.6 cells/ml for a final
culture volume of 100 to 250 ml.
[0140] The cells in suspension are collected and centrifuged for 10
min at 1 000 rpm approximately. The pellet is resuspended in 20 to
50 ml of 1.times.PBS (Phosphate buffer Salt). The cells are then
counted, centrifuged and the pelleted cells are taken up in a
serum-free medium at a final concentration of 3 to 5.times.10.sup.6
cells/ml. Several tubes are then prepared under these conditions
containing 3 to 5.times.10.sup.6 cells per tube.
[0141] Preparation of the Virus and Infection:
[0142] The viral stock having a known titer is rapidly thawed at
37.degree. C. and diluted in serum-free medium at a titer of
10.times. to 1 000.times. the concentration necessary for the final
infection. The cells are infected with the virus of interest at an
m.o.i. (multiplicity of infection) of 0.01 to 0.5 according to the
types of virus, which involves adding between 0.1 and 10%
volume/volume of viral suspension to the cellular pellet. After
incubating for 1 hour at an optimum temperature for the virus, in
general from 33 to 37.degree. C., the cells are again centrifuged
and the medium removed with care. This step is found to be often
necessary in order to limit the effect of the initial virus in the
subsequent process. One of the possibilities is to directly dilute
the cells without centrifuging them again with serum-containing
medium (5% of serum) at a final concentration of 0.2 to
1.times.10.sup.6 cells/ml and incubated again.
[0143] Harvesting of the Supernatant and of the Cells:
[0144] After 2 to 4 days of incubation, depending on the viral
kinetics and the potential cytopathic effect of certain viruses,
the medium containing the cells or the cellular debris is
harvested. Depending on the viruses, only the pellet or the
supernatant may be of interest and contain the viral particles. The
cells are harvested and centrifuged. The collected supernatant is
centrifuged again for 5 to 10 minutes at 2 500 rpm, and stored at
-80.degree. C. before purification of the particles. An aliquot is
collected in order to carry out the titration. The cellular pellet
is taken up in 5 ml of serum-free medium, sonicated and centrifuged
for 5 to 10 minutes at 2 500 rpm. The supernatant obtained is
stored at -80.degree. C. up to the purification and the titration
of an aliquot.
[0145] The viral infection and production efficiencies are compared
between the various conditions performed. For the viruses with
cytopathic effects, the titrations are in general carried out by
the lysis plaque technique.
EXAMPLE 14
Protocol for Infecting an Adherent Avian Cell Line (S86N45) with a
Virus
[0146] Preparation of the Cells:
[0147] The cells are inoculated 48 hours before the infection into
T150 flasks at a concentration of between 0.03 and 0.06 x 10.sup.6
cells/cm.sup.2 in a medium, preferably MacCoy's 5A, HAMF12 or DMEM
medium, or any other medium of interest, containing 5% serum. They
are maintained at 39.degree. C. and 7.5% CO.sub.2.
[0148] Infection:
[0149] The viral stock having a known titer is rapidly thawed at
37.degree. C. and diluted in serum-free medium at a titer of
10.times.to 1 000.times. the concentration necessary for the final
infection. The cells are infected with the virus of interest at an
m.o.i. (multiplicity of infection) of 0.01 to 0.5 according to the
types of virus, which involves adding between 0.1 and 10%
volume/volume of viral suspension to the cellular pellet. The
infection is generally carried out in a minimum of medium (from 5
to 10 ml for a 75 cm.sup.2 flask) in a medium containing 0%
serum.
[0150] After incubating for 1 hour at the optimum temperature for
the virus, in general from 33 to 37.degree. C., 20 ml of medium 5%
are added to the flasks. In a particular case, the cells can be
washed with PBS in order to remove the particles which might be
attached to the cells. In the case of a cytopathic virus, the cells
are observed daily after the infection in order to monitor the
appearance of the cell lysis plaque, which indicates good progress
of the infection.
[0151] Harvesting of the Supernatant and of the Cells:
[0152] After 2 to 4 days of incubation, depending on the viral
kinetics and the potential cytopathic effect of certain viruses,
the medium containing the supernatant, the cells and the cellular
debris are harvested. Depending on the viruses, only the pellet or
the supernatant may be of interest and contain the viral particles.
The cells are harvested and centrifuged. The collected supernatant
is centrifuged again for 5 to 10 minutes at 2 500 rpm, and stored
at -80.degree. C. before purification of the particles. An aliquot
is collected in order to carry out the titration. The cellular
pellet is taken up in 5 ml of serum-free medium, sonicated and
centrifuged for 5 to 10 minutes at 2 500 rpm. The supernatant
obtained is stored at -80.degree. C. up to the purification and the
titration of an aliquot.
[0153] The viral infection and production efficiencies are compared
between the various conditions performed. For the viruses with
cytopathic effect, the titrations are in general carried out by the
lysis plaque technique.
EXAMPLE 15
Replication of a Recombinant Avipox on Nonadherent Avian Stem Cells
of the EB1 Line
[0154] The nonadherent stem cells EB1 at passage 138 are amplified
and then infected at an m.o.i. of 0.1 with a recombinant avipox
producing a protein of interest. After the infection, the cells are
maintained in a spinner for the 4 days for which the infection
lasts. An aliquot is removed from the second day and then the next
two days in order to monitor the variation of the viral titer both
in the supernatant and in the intracellular content, after lysis of
the cellular content. The titration is carried out by the lysis
plaque technique.
[0155] Table 7 illustrates the results obtained. These results
demonstrate the very satisfactory replication of the recombinant
avipox on the EB1 stem cells. Thus, the infectious titer progresses
throughout the culture and the course of the infection, reaching a
maximum of 7.2 PFU/cell (PFU: Plating Forming Unit) after 4 days of
incubation. This titer is at least equivalent to that obtained for
this same recombinant avipox on primary chicken embryo cells.
[0156] This titer can be improved by specific culture conditions
and optimization procedures.
[0157] It will also be noted that at least equivalent infectious
titers were also obtained on a larger scale in 3 liter
bioreactors.
TABLE-US-00007 TABLE 7 Kinetics of titration of the recombinant
avipox on nonadherent EB1 stem cells Sampling (h after infection)
50 hours 74 hours 97 hours Cellular fraction 6.40 6.37 5.99 (Log
PFU/ml) Supernatant 5.56 5.8 6.29 (Log PFU/ml) Total 5.78 5.94 6.31
(Log PFU/ml) PFU/cell 2.2 3.2 7.2
EXAMPLE 16
Replication of Modified Vaccinia Virus Ankara (MVA) on Adherent and
Nonadherent Avian Stem Cells of the S86N45 Line and EB14 Line
[0158] The MVA virus (titer 2.5.times.107 TCID50/ml in 0.5 ml
vials) was received under frozen conditions.
[0159] For safety reasons, the MVA virus and infected cells were
kept under controlled conditions (-80.degree. C. freezer) and the
contaminated plastic material was placed into hypochloride solution
for more than 1 hour and then place into a bag for full and
complete autoclave inactivation.
[0160] Experiments and Results
[0161] The S86N45 and EB14 cells were thawed in a HamF12 based
complete medium.
[0162] The adherent S86N45 cells were amplified quite rapidly, with
a good growth rate and a nice morphology.
[0163] The cells were infected 1 hour in 2 ml of PBS with the
different m.o.i. of interest with no washing with PBS after the
infection. The medium was just added to the complete infectious
medium, i.e. the added virus was not removed.
[0164] After 3 days of infection, the cell lysis appears to be
proportional to the used m.o.i. This cytopathic effect is a good
indicator of the virus infection of the cells. So cells and
supernatant are harvested and stored at -80.degree. C. before
purification of particles and/or titration.
[0165] The non adherent EB14 cells were amplified. The cells were
infected, not washed, and the complete medium directly added on the
inocculum after 1 hour of contact with viral particles. After 3
days, a characteristic cell lysis was observed. The non infected
cells used as the control were counted and a good growth was
demonstrated, showing good culture conditions and therefore
confirming an efficient lysis by the virus in the infected culture.
Cells and supernatant are harvested and stored at -80.degree. C.
before purification of particles and/or titration (see table
8).
TABLE-US-00008 TABLE 8 Results of the titration Titer/mL Average
Virus/ based on Virus virus Cell pellet of 14 ml total yield/
yield/ Type M.O.I. Trial 10E6 cells volume cell cell S86N45 0 1 0 0
0 0 0 2 0 0 0 0.1 1 1-3 .times. 10{circumflex over ( )}7 .gtoreq.6
.times. 10{circumflex over ( )}5 5-15 5-15 0.1 2 1-3 .times.
10{circumflex over ( )}6 1-3 .times. 10{circumflex over ( )}5 1-5
0.01 1 1-3 .times. 10{circumflex over ( )}7 1-3 .times.
10{circumflex over ( )}6 15-30 .gtoreq.30 0.01 2 4-6 .times.
10{circumflex over ( )}7 1-3 .times. 10{circumflex over ( )}6
.gtoreq.30 EB14 0 1 0 0 0 0 0.1 1 1-3 .times. 10{circumflex over (
)}7 1-3 .times. 10{circumflex over ( )}6 15-30 15-30 0.1 2 1-3
.times. 10{circumflex over ( )}7 1-3 .times. 10{circumflex over (
)}6 15-30 0.05 1 .gtoreq.6 .times. 10{circumflex over ( )}7 4-6
.times. 10{circumflex over ( )}6 .gtoreq.50 .gtoreq.50 0.05 2 1-3
.times. 10{circumflex over ( )}7 1-3 .times. 10{circumflex over (
)}6 15-30 0.01 1 1-3 .times. 10{circumflex over ( )}7 1-3 .times.
10{circumflex over ( )}6 15-30 15-30 0.01 2 1-3 .times.
10{circumflex over ( )}7 1-3 .times. 10{circumflex over ( )}6
15-30
[0166] Applicants hereby incorporate by reference PCT/FRO3/00735
filed on Mar. 7, 2003, and French application serial no. 0202945
filed Mar. 8, 2002, and all references cited herein.
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