Note: Descriptions are shown in the official language in which they were submitted.
CA 022~6~67 1998-11-23
Process for culturing cells
The invention concerns a process for culturing mammalian
cells which require contact with cell surface proteins
for activation, differentiation and/or proliferation as
well as devices for culturing these cells.
The culture of human cells is of major importance for
various therapeutic approaches. Human cells cultured in
vitro are for example required in adoptive immunotherapy
with autologous or allogenic cells. Efficient processes
for culturing haematopoietic progenitor and stem cells
which for example can be transplanted into the patient
after radiation therapy or chemotherapy are also of
major importance.
Cytotoxic T lymphocytes (CTL) are responsible for
eliminating pathogenically changed endogenous cells such
as e.g. cells infected with viruses or tumour cells. In
adoptive immunotherapy, lymphocytes of the patient are
activated in vitro and then reimplanted. Such an
activation can for example be carried out by adding
interleukin 2 (IL2) to promiscuous killer cells (Thiele,
D. et al., Immunology Today 10 (1989) 375 - 381). Such
cells are then referred to as lymphokine-activated
killer cells (LAK cells) (Rosenberg, Immunology Today 9
(1988) 58 - 62). However, LAK cells are also obtained in
the stimulation with IL2 which are directed against
healthy endogenous cells (Chen, B. et al., Cell Immunol.
118 (1989) 458 - 469). In a further method for adoptive
immunotherapy, the lymphocytes to be activated are
CA 022~6~67 1998-11-23
cultured in the presence of autologous tumour cells
(Mixed Lymphocyte Tumor Cultures, Fossati, G. et al.,
International Journal of Cancer, 42 (1988) 239 - 245). A
further method is described in WO 94/23014. According to
this method lymphocytes are activated to form
tumoricidal cells in a co-culture with a mammalian cell
line while avoiding an allogenic stimulation. Fragments
or vesicles of this cell line can also be used instead
of the cell line described in this reference.
Suspended vital tumour cells or fragments thereof which
have been previously advantageously inactivated by
chemotherapy or radiotherapy are usually used for the ex
vivo activation of CTLs. However, this process has major
disadvantages. The inactivation of the tumour cells is
complicated (irradiation, handling of toxic substances).
It cannot be excluded that vital tumour cells or DNA
from tumour cells are carried over into the transplant
during transplantation.
Furthermore the inactivation of cells can lead to
receptor modulations. Also the secretion of inhibitory
molecules by for example inactivated tumour cells that
are still alive cannot be excluded. Also the geometric/
mechanical problem of the optimal cell density of
effector to activator cells (probability of hits) is
time-consuming and can only be determined empirically.
The immobilization of biological effectors on cell
culture surfaces is used to activate cells and to
proliferate them. Thus for example anti-T-cell
antibodies are immobilized by preincubation on cell
culture vessels by means of non-covalent binding. T
cells that are added proliferate by binding/interaction
CA 022~6~67 1998-11-23
of their CD3 receptors with immobilized (CD3) antibodies
(Geppert, T.D., Lipsky, P.E., The Journal of Immunology
6, Vol. 138 (1987) 1660-1666).
Antigen-specific CTL's can be induced in a similar
process by immobilizing MHC molecules alone (Walden, P.,
et al., Nature, Vol. 315 (1985) 327-329) or embedded in
synthetic, planar membranes (Watts, T.H. et al., Proc.
Natl. Acad. Sci. USA, Vol. 81 (1984) 7564-7568).
Moreover the T cell activation can be modulated by
interactions with immobilized accessory molecules (Moy,
V.T., Brian, A.A., J. Exp. Med. 175 (1992) 1-7).
Furthermore cells can be immobilized by non-covalent
binding on cell culture vessel surfaces. The binding of
monocytes on FCS-coated culture vessels and pulsing with
specific antigens leads, after co-culture with
peripheral blood lymphocytes, to an improvement of the
antigen presentation with increased antibody production
of the B cells (Jahn, S., et al., Allerg. Immunol. 33
(1987) 239-244).
The preparation of artificial lipid vesicles is state of
the art. These liposomes can, on the one hand, be loaded
with proteins embedded in the lipid membrane to improve
cell targeting in vitro (Herrmann, S.H., Mescher, M.F.,
Proc. Natl. Acad. Sci. USA, Vol. 78, No. 4 (1981) 2488-
2492; Bloemen, P.G.M., et al., FEBS Letters 357 (1995)
140-144; Bergers, J.J., et al., Journal of Controlled
Release 29 (1994) 317-327; Gregoriadis, G., Immunology
Today, Vol. 11, No. 3 (1990) 89-97). On the other hand,
chemotherapeutic agents can be enclosed in such vesicles
to increase the local dose in order to induce cell death
(Brown, P.M., Silvius, J.R., Biochimica et Biophysica
CA 022~6~67 1998-11-23
Acta 1023 (1990) 341-351). The in vivo administration of
artificial anti-tumour vesicles tliposomes) for antigen
immunotherapy also corresponds to the state of the art
(Phillips, N.C., et al., Liposomes in the Therapy of
Infectious Diseases and Cancer, 1989, Alan R. Liss, Inc.
(ed.), p. 15-24; Bergers, J.J., et al., Cancer Immunol.
Immunother. 34 (1992) 233-240; Papahadjopoulos, D.,
Gabizon, A., Annals of the New York Academy of Sciences,
Vol. 507, R.L. Juliane (ed.), 1987, 64-74).
In the previously known processes artificial liposomes/
vesicles are either used in solution by non-covalent
binding to carriers or in vivo. It is also known that
cells labelled covalently via a binding partner can be
used in a cell separation process to enable separation
of bound specific cells from undesired cells by means of
immobilized antibodies (EP-A 0 701 130).
A process for culturing haematopoietic progenitor stem
cells using feeder layers of stroma cells is described
in W0 95/02040. However, the preparation of such feeder
layers is time-consuming.
The object of the invention is to provide an improved
process for culturing mammalian cells which contact cell
surface proteins of other cells during culture.
This object is achieved by a process for culturing a
first mammalian cell which contacts cell surface
proteins of a second mammalian cell for culturing
characterized in that the said first mammalian cell is
cultured in the presence of an immobilized vesicle of
the second mammalian cell which contains parts of the
natural surface of the second mammalian cell.
CA 022~6~67 1998-11-23
Instead of a vesicle of a second mammalian cell it is
also possible to use a complete second mammalian cell.
In this case a second mammalian cell is used which is
modified with a first partner of a biological binding
pair and is bound to a solid carrier via the second
partner of the biological binding pair.
It is expedient to immobilize the cells or membrane
vesicles by incubation with a bindable carrier. The
binding is achieved by means of a biological binding
pair such as for example streptavidin/avidin,
sugar/lectin, a CD molecule (e.g. CD3/anti-CD3
antibody), MHC molecules tclass I or II) or
digoxigenin/anti-digoxigenin antibodies.
The use of the binding partners streptavidin/biotin,
avidin/biotin or derivatives of these compounds for the
immobilization of cells or vesicles thereof has not been
previously described. The preparation of biotinylated
artificial liposomes in which biotin is bound to lipids
was previously known (Bayer, E.A., Wilchek, M., Liposome
technology, CRC Press Inc., 1984, Vol. III, 127-135;
Loughrey, H.C., et al., FEBS 13102, Vol. 332, No. 1,2
(1993) 183-188). The characteristics of their binding to
SA-labelled microtitre plates were examined and the
primary aim was to bind these artificial biotin vesicles
in vivo to SA-labelled cells (Corley, P., Loughrey,
H.C., Biochimica et Biophysica Acta 1195 (1994) 149-
156).
It has turned out that numerous simultaneously active
interactions between cells, antigens and/or factors are
necessary for the activation and differentiation of
cells by other cellular binding partners (Herold, C., et
CA 022~6~67 1998-11-23
al., MS-Medecine Sciences, Vol. 11, No. 5 (1995) 669-
680; Clark, E.A. Ledbetter, J.A., Nature 367 (1994) 425-
428; Mayordomo, J.I., et al., Nature Medicine, Vol. 1,
No. 12 (1995) 1297-1302). Therefore the use of
preferably biotinylated immobilized cells or native
membrane vesicles derived therefrom is a major advantage
since, surprisingly all necessary reaction partners are
located on or in the membrane. Moreover such an
artificial surface is a universal reaction system since
partners that may be absent, e.g. in the case of mutated
tumour cells, can be easily additionally applied by
means of biological binding pairs.
Mammalian cells which are suitable for culture by the
process according to the invention are cells which for
their culture make a receptor and/or antigen contact
with cell surface proteins of other mammalian cells.
Hence bioequivalent cell surfaces are required for
culture. Such surface proteins are for example MHC
complexes, co-stimulatory signals or adhesion molecules
(Mescher, M.F., Immunological Reviews 146 (1995) 177;
Herold, C., et al., MS-Medecine Sciences, Vol. 11, No. 5
(1995) 669-680; June, C.H., et al., Immunology Today 15
(1994) 321).
The required complexity of the interactions of cell
surface proteins for cell activation, cell
differentiation and cell proliferation become
particularly apparent when co-culturing bone marrow
stroma cells with haematopoietic stem cells for the
long-term culture of the latter cells (Sutherland, H.J.
Eaves, C.J., Culture of Hematopoietic Cells, Freshney,
R.J., et al., (eds.), Wiley Liss, N.Y., 1994, pp. 139;
Koller, M.R., et al., Biotechnology 11 (1993) 358). This
seems to also apply to the differentiation of T cells
CA 022~6~67 1998-11-23
from haematopoietic precursor cells ex vivo where co-
culture with foetal thymus cells/tissue is required
(Dou, Y.M., et al., Thymus 23 (1994) 195).
The culture of the mammalian cell can according to the
invention be an expansion, an enrichment of a particular
sort of cell from a cell mixture, an activation,
differentiation and/or a proliferation of a mammalian
cell. The process can be applied particularly
advantageously to the proliferation of cytotoxic T cells
and the differentiation and proliferation of
haematopoietic progenitor and stem cells in
granulocytes, monocytes, erythrocytes, megakaryocytes
and lymphocytes.
In a preferred embodiment the cultured cells are
modified by genetic engineering during the culture for
example by transfection with a vector or by transduction
with a therapeutically relevant retrovirus (Einerhand,
M.P.W., et al., Blood 81 (1993) 254; Nolta, J.A., et
al., J. Clin. Invest. 90 (1992) 342). Such ex vivo
genetically modified cells can be used for a gene
therapy.
In addition to intact cells, vesicles or fragments of
cells are also suitable for co-culture to which a
binding partner of a biological binding pair is bound.
Cells that are suitable as cells for preparing such
fragments or vesicles are those which are usually used
for the co-culture of mammalian cells. These are for
example stroma cells as a feeder layer, thymus cells,
antigen-presenting cells or other cells which carry the
signals for a T cell activation. Such signals are
essentially those which mediate cell contacts such as
CA 022~6~67 1998-11-23
e.g. adhesion (for example via CDlla, CD18, CD54),
antigen-specific recognition (by means of the MHC
complex via T cell receptor) or co-stimulating signals
(for example via B7/CD28). This can also include the
presentation of growth factors (Toksoz, D., et al.,
Proc. Natl. Acad. Sci. USA 89 (1992) 7350).
Cell vesicles can be prepared by methods familiar to a
person skilled in the art. Vesicles can for example be
prepared by hypotonic shock or by incubation with
cytochalasin B. Other methods are for example described
in WO 94/23014. Vesicles are obtained by such production
methods which present parts of the native cell surface
of the starting cell to other cells but are themselves
no longer capable of replication. Consequently such
vesicles no longer contain a cell nucleus. The cell
nuclei are preferably separated from the vesicles by
filtration (ca. 2-5 ~m pore diameter). If it is intended
to use vesicles which are modified with a binding
partner of a biological binding pair it is expedient to
modify the intact cells and subsequently to form
vesicles from these cells.
Binding of a binding partner of the biotin/streptavidin
system or biotin/avidin system, preferably biotin, is
carried out expediently by biotinylating the whole
cells. In this process free amino groups of proteins
located on the cell membrane are preferably bound to
biotin. A suitable reagent for this is for example D-
biotinoyl-y-aminocarboxylic acid-N-hydroxysuccinimide
ester (biotin-7-NHS).
Whether and to what extent the cells or vesicles have to
be further modified before immobilization (binding of a
.
CA 022~6~67 1998-11-23
binding partner of the biological system to the surface)
essentially depends on the binding pair that is used. If
for example the binding pair CD3/anti-CD3 antibody or
lectin/sugar is used then it is not necessary to modify
the cells or vesicles before immobilization. In this
case it is sufficient if the carrier is for example
coated with anti-CD3 antibody or lectin. The cells or
vesicles can then bind via structures (CD3 or sugar)
that are naturally present on their surface to the
corresponding binding partner which is immobilized on
the carrier. A further advantage of such a process is
that defined vesicles can be selectively bound. For
example vesicles can be prepared from a PBMNC
preparation and only bind T cells (CD3+) via an anti-CD3
antibody. A similar differentiation of vesicles can also
be accomplished with other suitable surface markers. In
addition to a binding pair in which one binding partner
occurs naturally on the surface of the cells or vesicles
to be immobilized, it is also possible to use a binding
pair where neither of the binding partners occurs
naturally on the surface of cells or vesicles. In this
case the cells or vesicles must be modified before
immobilization. Preferably the partner of the biological
system that is to be bound is bound covalently to the
cell surface via an activated amino group. Such binding
pairs are for example biotin/streptavidin,
digoxigenin/anti-digoxigenin antibody. In this case it
is preferable to use a binding pair whose affinity is
very high.
In this process it is not necessary that vesicle and
carrier each carry on their surface the respective
opposite partner of the biological binding pair, but
rather it is also possible that the vesicle and carrier
carry the same binding partner on the surface. In this
CA 022~6~67 1998-11-23
-- 10 --
case the other binding partner must be added, preferably
in soluble form for the immobilization. Thus for example
the vesicle and carrier surface can be biotinylated and
the immobilization is achieved by adding soluble
streptavidin. It is also possible to bind CD3 protein to
the surface of the carrier and to immobilize vesicles
which carry CD3 protein on the surface by adding a
soluble antibody which is directed against CD3.
In a further embodiment the vesicles can be modified
with more than one binding partner. In this case they
are bound to the carrier via a binding partner whereas
other substances can be bound to the surface of the
cells or vesicles by means of the other binding partner.
Substances are preferably bound which are required for
the activation, proliferation or differentiation of
cell. Antigens are preferably used which can increase
the immunogenicity of tumour cells (activation antigens
such as B7, CD40, MHC II or ICAM).
However, it is also possible that the tumour cell
vesicles lack essential signal-triggering surface
proteins. Such surface proteins are normally either
naturally present on the normal cells of tumour patients
or are inducible. Such signal proteins are for example
the co-stimulatory signal proteins of the B7 family or
CD40, antigen-presenting receptors such as MHC I and/or
MHC II, adhesion molecules such as ICAM-1 or LFA-3 which
are usually provided by normal haematopoietic cells.
Therefore in a preferred embodiment of the invention a
mixture of vesicles of different cell populations is
used, preferably a mixture which contains tumour cell
vesicles as well as other vesicles which carry co-
CA 022~6~67 1998-11-23
-- 11 --
stimulatory signal-triggering surface proteins. Such
other vesicles are preferably prepared from
haematopoietic cells, from monocytes, macrophages,
dendritic cells or B cells. Such cells can quite
generally be used to prepare other vesicles which
naturally carry or provide the missing signals or induce
the missing signals. In this process hybrid surfaces of
mixed immobilized vesicles are for example formed from
tumour cells and from normal cells. In this manner it is
for example possible to induce effective CTLs in the co-
culture of for example autologous T cells of tumour
patients.
The cells which are selected to be used for the
preparation of vesicles with signal peptides can be
directly isolated or purified from body fluids or tissue
such as blood and/or bone marrow. They can also be grown
in vitro by suitable culture conditions. When mixed
hybrid vesicle surfaces are prepared, the same amounts
of the respective vesicles are preferably immobilized.
Depending on the vesicle size and density of the
vesicles to be immobilized it is, however, also possible
to immobilize relative proportions which differ from
this.
In a further embodiment the mammalian cells that are to
be cultured are firstly contacted in a first step with a
first sort of vesicles and, in a further culture step,
are contacted with a further sort of vesicles. This is
advantageously carried out until an adequate stimulation
is achieved.
The vesicles preferably have a size of about 10 - 30 %
of the first mammalian cell to be cultured. If it is
CA 022~6~67 1998-11-23
intended to co-culture with vesicles from different
cells, these vesicles preferably have a size which
enables a simultaneous contact with at least two
different vesicles. With a cell size of ca. 10 ~m, the
vesicles are preferably below 500 nm, preferably below
100 nm in diameter.
In a further embodiment such activating substances can
also be bound to the surface of cells or vesicles if
these are only modified with a binding partner. In this
case the cells or vesicles are immobilized on the carrier
via the binding partner in a first step and in a second
step the activating substances are bound to the binding
partners that are still freely available on the surface
of the cells or vesicles. In the case of biotin as the
binding partner, the cell or the vesicle is consequently
firstly bound to the carrier via biotin/streptavidin
interaction and subsequently streptavidin-bound B7 is
added to the biotins that are still free on the surface
of the cells or vesicles, or biotinylated B7 and soluble
streptavidin is added.
Antibodies to MHC class I or MHC class II molecules can
for example also be used as binding partners for cell
surface receptors (if unmodified vesicles are used).
Binding partners preferably on carriers coated with
streptavidin or avidin. SUCh an incubation for
immobilization can be carried out in a simple manner for
several hours at room temperature or at an elevated
temperature. Subsequently the non-immobilized fraction
of the cells or vesicles is removed by washing.
In a preferred embodiment tumour cells, vesicles of
tumour cells or lymphocytes isolated by means of a
.
CA 022~6~67 1998-11-23
Ficoll gradient are used as second mammalian cells for
co-culturing.
An advantage of the process according to the invention
is that defined vesicles can be used for example in
stromal two step cultures since they can be combined
with different cytokines or ECM proteins and hence the
culture conditions can be varied as desired. This
results in a culture preparation that can be more
readily controlled.
Therefore in a preferred embodiment growth factors such
as e.g. SCF, IL1, IL-3, IL-6, IL-11, EPO, G-CSF, GM-CSF,
flt-2/flk-3 etc. can be added during the co-culture.
In a further preferred embodiment such growth factors or
factors which mediate a co-stimulatory signal or an
adhesion signal can be bound to streptavidin when the
cell surface is biotinylated and thus be bound to the
cell surface. This is particularly advantageous when
vesicles of tumour cells are used which do not usually
carry a co-stimulatory signal. In this case it is
particularly preferable to for example bind
streptavidin-bound B7 to the cell surface. This can
increase the immune reactivity of tumour cells.
The following examples, publications and figures further
elucidate the invention the protective scope of which
results from the patent claims. The described processes
are to be understood as examples which still describe
the subject matter of the invention even after
modifications.
.. .. -- . . . . . . .. .
CA 022~6~67 1998-11-23
- 14 -
Fig. 1 shows the homogeneous coating of streptavidin-
coated cell culture plates (96-well plates) with
biotinylated vesicles of L88/5 bone marrow
stroma cells (100 x enlargement).
Fig. 2 shows the flow-cytometric analysis of cells and
isolated vesicles
A-C: intact L88/5 bone marrow stroma cells
D-F: isolated vesicles of L88/5 bone marrow
stroma cells.
A/D: morphological analysis of particles by
forward and side scattered light (FSC vs. SSC)
B/F: Detection of cell-bound or vesicle-bound biotin
on biotinylated L88/5 cells by means of FITC-
labelled streptavidin (F11). Dead cells or
vesicles of dead cells are labelled with
propidium iodide (F12).
C/F: as B/F only on non-biotinylated L88/5 cells.
Examp 1 e
Culture of ctroma cells
The stromal cell lines L87/4 and L88/5 were cultured in
long-term medium as described by Thalmeier et al., Blood
83 (1994) 1799-1807 and Wo 95/02040. The stromal cell
lines L87/4 and L88/5 were cultured as one layer
cultures in plastic culture flasks (NUNC, Wiesbaden-
Biebrich) using LTC medium containing 10-6 M
hydrocortisone (HSS). The cells were incubated at 370C
in a Co2 incubator (air/5 % C02), trypsinized and
subcultured at an initial concentration of 3 x 105
cells/25 cm2 culture flask.
CA 022~6~67 l998-ll-23
Long-term medium:
McCoy's 5a containing 12.5 % FCS; 12.5 % HS; 1 ~ sodium
bicarbonate; 1 % sodium pyruvate; 1 % vitamins; 0.4 ~
non-essential amino acids; 0.8 % essential amino acids;
200 mM L-glutamine; 1 ~ penicillin/streptomycin; 10-4 M
a-thioglycerol; 10-6 M hydrocortisone.
Example 2
Isolation of the stroma cell suspension
After reaching confluence the cells are detached with
0.25 % trypsin (Gibco) (10 min; 37~C), the cell count is
determined and they are washed in PBS (without Mg2+ and
Ca2+) (10 min; 1200 rpm; RT). The cell pellets (ca. 2-3 x
107 cells in the case of L87/4; ca. 5 x 107 cells in the
case of L88/5) are resuspended in 1 ml PBS each time.
Example 3
Biotinylation of the stromal cells
In order to label the cell membranes with biotin, 10 ~l
biotin-7-NHS solution (D-biotinoyl-~-aminocaproic acid-
N-hydroxysuccinimide ester; Boehringer) is added each
time and shaken for 10 min at RT. Free amino groups of
the proteins located at or on the cell membrane react
with biotin-7-NHS with formation of a stable amide bond.
Non-reacted biotin-7-NHS is removed by washing twice
with 50 ml PBS each time.
Biotin-7-NHS solution:
5 mg biotin-7-NHS in 250 ~l DMS0 (= 20 mg/ml).
.
CA 022~6~67 l998-ll-23
-- 16 --
Example 4
Preparation of vesicles ~modified according to Jett et
al., J. Biol. Chem. 252 (1977) 2134-2143)
The biotinylated cells are resuspended in 1 ml aliquots
of pre-heated EBSS (Sigma). Subsequently a 90 ~ glycerol
solution (37~C) is added by pipette in 3 equal portions
at 5 minute intervals to the cell suspension in order to
finally obtain a 30 % glycerol solution. All steps are
carried out at 37~C. 5 min after the last glycerol
addition the cell suspension is cooled for 2 min in an
icebath. All further steps are carried out at 4~C. The
cells are centrifuged (10 min. 1200 rpm; 4~C) and the
supernatant is discarded. The cells are burst (hypotonic
shock) by the rapid addition of 1 ml cold Tris buffer
and briefly vortexed. After 5 min incubation in the
icebath the lysed cells are centrifuged (10 min; 1000
rpm; 4~C). The lysate (= supernatant) is filtered with
the aid of a 5 ~m filter unit (Millex~-SV filter unit;
Millipore). The pelleted cell debris is in turn
resuspended in 1 ml cold Tris buffer, vortexed and also
filtered through a 5 ~m filter unit. As a result the
larger membrane particles and the cell nuclei are
separated and a homogeneous suspension is obtained
composed of (< 2 ~m) membrane vesicles of approximately
equal size. The filtrates are combined and centrifuged
(20 min; 6700 x g; 4~C).
Glycerol solution:
go ~ w/v Earle's balanced salt solution (EBSS).
Tris buffer:
10 mM Tris-Cl, pH 7.4; 1 mM MgCl2; 1 mM CaCl2.
CA 022~6~67 l998-ll-23
-- 17 --
Example S
Immobilization of membrane vesicles by means of a
biotin-streptavidin binding
The pelleted membrane vesicles are taken up in ca. 5 ml
medium and plated out in an amount of 50 ~l/well in
streptavidin-coated 96-well plates. After an incubation
period of 5-10 hours at 37~C in a CO2 incubator (air/5 %
C02) the non-bound vesicles can be removed by washing.
Plate technology:
The cell culture plates (sterile) coated with
streptavidin can be very homogeneously coated with the
biotinylated membrane vesicles (< 2 ~m) i.e. with very
small and relatively uniform spaces between them. The
vesicles adhere to the plates via the biotin-
streptavidin binding after 5-10 hours incubation period
and remain as a uniform coating even after vigorous
shaking or rinsing (Fig. 1).
The coating of the plates results in a homogeneous
vesicle lawn which could be stored for at least one
week.
Analysis of the vesicles:
In order to examine whether the prepared vesicles were
also actually labelled with biotin and how uniformally
this labelling occurred, the cells and vesicles were
analysed at various times during the preparation. Fig.
2B clearly shows that the majority of the cells are
biotinylated (right lower cluster) after labelling with
FITC-conjugated streptavidin. The control preparation of
non-biotinylated cells is shown in Fig. 2C. The prepared
vesicles (Fig. 2E) are all biotinylated. The control
, ~ _ ... .. . . . . ...
CA 022~6~67 l998-ll-23
-- 18 --
preparation of non-biotinylated vesicles is shown in
Fig. 2F.
Even after an incubation period of at least one week at
37OC, the individual vesicles were still clearly
recognizable in the streptavidin plates and still bound
to the plates. Equally the vesicles could be stored for
a period of at least 8 days at 4~C in medium or buffer
without reducing their ability to attach via the biotin-
streptavidin binding. The vesicles could be used in a
comparable manner to freshly prepared vesicles after
brief vortexing.
Example 6
Co-culture of human CD34+ cells from umbilical cord
venous blood or bone marrow on vesicle-coated 96-well
plates compared to irradiated cell layers or plastic
Isolation of CD34+ cells:
The method used here is the immunomagnetic separation of
CD34+ cells from mononuclear cells (MNC) with the aid of
Dynabeads~ (Deutsche Dynal GmbH, Hamburg). For this the
precursor cells were labelled with paramagnetic beads
covalently bound to CD34 antibodies, separated from the
unlabelled cells in a magnetic field and subsequently
the beads were detached from the cells with the aid of a
specific antibody preparation (DETACHaBEAD~, Deutsche
Dynal GmbH). Umbilical cord venous blood was diluted 1:5
with RPMI (containing 25 units/ml heparin and 10 ~g/ml
DNase) in order to ensure the most efficient possible
yield of MNC. The cell suspension was layered on Ficoll-
Paque~ and centrifuged for 30 min at RT and 1600 rpm.
Subsequently the cells at the interphase were washed
CA 022~6~67 1998-11-23
-- 19 --
twice (RPMI/heparin/DNase), the cells were taken up in
RPMI/10% FCS + DNase and the cell count was determined.
The M-450 CD34 beads were washed with RPMI/10 % FCS
before use and well-mixed with the cells in Eppendorf
vessels. The optimal ratiQ of beads to cells was 1:1.
The cell bead mixture was incubated for 45 min at 4~C
while gently rotating. Subsequently the labelled cells
were separated with the aid of a Dynal MPC magnet by
washing four times and taken up in 100 ~l RPMI/10 % FCS.
In order to detach the beads from the cells, the cell
suspension was well-mixed with one unit (10 ~l)
DETACHaBEAD~ per 107 Dynabeads and incubated for 60 min
at RT while gently rotating. The detached CD34+ cells
were separated from the beads with the aid of a magnet
by washing twice. Subsequently the cell count was
determined and the precursor cells were subjected to the
various tests.
DNase sol ution:
dissolve 10 mg DNAase in 1 ml PBS; filter sterile
Two step cultures:
The stromal cell lines were sown at a cell density of
104/well in LTC medium containing 10-6M HSS in 96-well
plates, incubated for 24 hours at 37~C in an incubator,
irradiated with 15 or 20 gray (caesium 137, gamma cell
irradiation apparatus) and cultured for a further 12-24
hours. Then the CD34+ precursor cells isolated from
umbilical cord venous blood (3.2.10.1 or 3.2.10.2) were
sown on the various cell layers (1-2 x 103 CD34+ cells/
well) or on immobilized vesicles. Half of the consumed
medium was removed once per week and replaced by fresh
medium. After the end of the culture period (in this
CA 022~6~67 1998-11-23
- 20 -
case 2 weeks) all cells were harvested, the cell count
was determined and they were used in methylcellulose
cultures.
Methylcellulose culture:
In order to quantify the clonality of CD34+ cells which
had been cultured in various vesicle-coated 96-well
plates or on various stromal layers, methylcellulose
cultures were prepared. For this semisolid cultures were
set up containing 104 to 2 x 104 non-adherent cells
per ml in 14 % IMDM, 30 % FCS, 1 % BSA, 5 % PHA-LCM,
10-4 M each of L-glutamine and ~-thioglycerol, 0.98 %
methylcellulose, 3 units/ml EPO and 100 ng/ml KL. These
cultures were incubated at 37~C in 1 ml volume in 35 mm
tissue culture plates (air/5 % CO2) and the colonies
were counted after 14 days with the aid of an invert
microscope (Zeiss).
Results of clonal growth:
The number of GM-CFC in the co-culture on L87/4 or L88/5
was 465.5 or 116.3 clones. In the co-culture on
corresponding vesicles 90 and 76.3 GM-CFC clones
respectively grew. The number of BFU-E in the co-culture
of L87/4 or L88/5 was 104.5 or 13.8 clones respectively.
In the co-culture on respective vesicles 27.5 or 52.5
BFU clones respectively grew. The co-culture of CD34
cells on vesicles of the stroma cell line L87/4 compared
to the plastic surface alone of cell culture vessels
resulted in 21.3 GM-CFC and 640 BFU-E clones in the
vesicle co-culture and 16.3 GM-CFC and 217.5 BFU-E
clones in the culture on a plastic surface.
CA 022~6~67 l998-ll-23
-- 21 --
Li~t of References
Bayer, E.A., Wilchek, M., Liposome Technology, CRC
Press, Inc. 1984, Vol. III, 127-135
Bergers, J.J., et al., Cancer Immunol. Immunother. 34
(1992) 233-240
Bergers, J.J., et al., Journal of Controlled Release 29
(1994) 317-327
Bloemen, P.G.M., et al., FEBS Letters 357 (1995) 140-144
Brown, P.M., Silvius, J.R., Biochimica et Biophysica
Acta 1023 (1990) 341-351
Chen, B., et al., Cell Immunol. 118 (1989) 458-469
Clark, E.A., Ledbetter, J.A., Nature 367 (1994) 425-428
Corley, O., Loughrey, H.C. Biochimica et Biophysica Acta
1195 (1994) 149-156
Dou, Y.M., et al., Thymus 23 (1994) 195
Einerhand, M.P.W., et al., Blood 81 (1993) 254
EP-A 0 701 130
Fossati, G. et al., International Journal of Cancer, 42
(1988) 239-245
Geppert, T.D., Lipsky, P.E., The Journal of Immunology
6, Vol. 138 (1987) 1660-1666
Gregoriadis, G., Immunology Today, Vol. 11, No. 3 (1990)
89-97
Herold, C., et al., MS-Medecine Sciences, Vol. 11, No. 5
(1995) 669-680
Herrmann, S.H., Mescher, M.F., Proc. Natl. Acad. Sci.
USA, Vol. 78, No. 4 (1981) 2488-2492
Jahn, S., et al., Allerg. Immunol. 33 (1987) 239-244
Jett et al., J. Biol. Chem. 252 (1977) 2134-2143
June, C.H., et al., Immunology Today 15 (1994) 321
Koller, M.R., et al., Biotechnology 11 (1993~ 358
Loughrey, H.C. et al., FEBS 13102, Vol. 332, No. 1,2
(1993) 183-188
Mayordomo, J.I., et al., Nature Medicine, Vol. 1, No. 12
CA 022~6~67 l998-ll-23
- 22 -
(1995) 1297 - 1302
Mescher, M.F., Immunological Reviews 146 (1995) 177
Moy, V.T., Brian, A.A., J. Exp. Med. 175 (1992) 1-7
Nolta, J.A., et al., J. Clin. Invest. 90 (1992) 342
Papahadjopoulos, D., Gabizon, A., Annals of the New York
Academy of Sciences, Vol. 507, R.L. Juliane (ed.),
1987, 64-74
Phillips, N.C., et al., Liposomes in the Therapy of
Infectious Diseases and Cancer, 1989, Alan R. Liss,
Inc. (ed.), p. 15-24
Rosenberg, Immunology Today 9 (1988) 58-62
Sutherland, H.J., Eaves, C.J., Culture of Hematopoietic
Cells, Freshney, R.J., et al., (eds.), Wiley Liss,
N.Y., 1994, pp. 139
Thalmeier et al., Blood 83 (1994) 1799-1807
Thiele, D., et al., Immunology Today 10 (1989) 375-381
Toksoz, D., et al., Proc. Natl. Acad. Sci. USA 89 (1992)
7350
Walden, P., et al., Nature, Vol. 315 (1985) 327-329
Watts, T.H., et al., Proc. Natl. Acad. Sci. USA, Vol. 81
(1984) 7564-7568
W0 94/23014
W0 95/ 02040
