U.S. patent application number 14/351889 was filed with the patent office on 2014-10-23 for method and device for cell modification.
This patent application is currently assigned to MILTENYI BIOTEC GMBH. The applicant listed for this patent is Miltenyi Biotec GmbH. Invention is credited to Stefan Miltenyi, Alexander Scheffold.
Application Number | 20140315311 14/351889 |
Document ID | / |
Family ID | 47148825 |
Filed Date | 2014-10-23 |
United States Patent
Application |
20140315311 |
Kind Code |
A1 |
Miltenyi; Stefan ; et
al. |
October 23, 2014 |
Method and device for cell modification
Abstract
The invention relates to a cell modification device, comprising
a centrifugation chamber with at least one cell modifying surface
with a normal vector having an angle of 135-45.degree. to the
rotational axis of the centrifugation chamber, wherein the
centrifugation chamber comprises at least one input/output port and
the cells to be modified are immobilized at the cell modifying
surfaces by the rotation of the centrifugation chamber at 2 to 2000
g. Furthermore, the invention relates to a method for modifying
cells comprising the steps--introducing cells in a cell
modification device, comprising a centrifugation chamber with at
least one cell modifying surface with a normal vector having an
angle of 135-45.degree. to the rotational axis of the
centrifugation chamber wherein and comprising at least one
input/output port, --immobilizing the cells on the cell modifying
surfaces by the rotation of the centrifugation chamber at 2 to 2000
g--maintaining the rotation of the rotation of the centrifugation
chamber until the cells are modified.
Inventors: |
Miltenyi; Stefan; (Bergish
Gladbach, DE) ; Scheffold; Alexander; (Cologne,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Miltenyi Biotec GmbH |
Bergisch Gladbach |
|
DE |
|
|
Assignee: |
MILTENYI BIOTEC GMBH
Bergisch Gladbach
DE
|
Family ID: |
47148825 |
Appl. No.: |
14/351889 |
Filed: |
November 13, 2012 |
PCT Filed: |
November 13, 2012 |
PCT NO: |
PCT/EP2012/072431 |
371 Date: |
April 15, 2014 |
Current U.S.
Class: |
435/455 ;
435/285.1; 435/325 |
Current CPC
Class: |
C12N 2510/00 20130101;
C12M 35/04 20130101; C12M 23/20 20130101; C12M 35/08 20130101; C12N
2525/00 20130101; C12M 41/48 20130101; C12N 5/0636 20130101; C12M
27/10 20130101; G16B 20/00 20190201; C12N 15/85 20130101; C12N
15/87 20130101; C12M 45/05 20130101 |
Class at
Publication: |
435/455 ;
435/325; 435/285.1 |
International
Class: |
C12N 15/85 20060101
C12N015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 18, 2011 |
EP |
11189754.2 |
Claims
1. A cell modification device, comprising a centrifugation chamber
with at least one cell modifying surface with a normal vector
having an angle of 135-45.degree. to the rotational axis of the
centrifugation chamber, wherein the centrifugation chamber
comprises at least one input/output port and the cells to be
modified are immobilized at the cell modifying surfaces by the
rotation of the centrifugation chamber at 2 to 2000 g and wherein
the cell modifying surfaces are selected from the group of cell
modifying surfaces that are functionalized and those that can be
functionalized for cell modification.
2. A cell modification device according to claim 1, wherein the
cell modifying surface is functionalized with at least one
substance wherein the substance is selected from the group of
substances that enhance proliferation of cells, that induce genetic
modification and that induce cellular modification of cells.
3. A cell modification device according to claim 1, wherein the
cell modifying surface is functionalized with particles being
functionalized with at least one substance selected from substances
that enhance proliferation of cells, induce genetic modification,
and induce cellular modification of cells.
4. A cell modification device according to claim 2, wherein the
substance enhancing proliferation of cells is selected from the
group consisting of collagen types (I to VIII), fibronectin,
gelatin, laminin, elastin, hyaluronic acid, keratan sulfate,
chondroitin sulfate, heparin sulfate proteoglycans, poly-d-lysine,
avidin, streptavidin, biotin, antibodies, antibodies against biotin
or protein tags, protein tags like IIsopeptag, BCCP, Myc-tag,
Calmodulin-tag, FLAG-tag, HA-tag, His-tag, Maltose binding
protein-tag, Nus-tag, Glutathione-S-transferase-tag, Green
fluorescent protein-tag, Thioredoxin-tag, S-tag, Softag 1, Softag
3, Strep-tag, SBP-tag, Ty tag, certia, poly lactate, polyvinyl
alcohols, polysaccharides and dextran.
5. A cell modification device according to claim 2, wherein the
substance inducing cellular modification of cells is selected from
the group consisting of agonistic or antagonistic antibodies,
cytokines, growth factors, (de-)activating ligands,
pharmacologically active substances, mitogens, DNA or RNA-modifying
substances.
6. A cell modification device according to claim 2, wherein the
substance inducing genetic modification of cells is selected from
the group consisting of a virus, viral particle, adenovirus,
retrovirus, lentivirus, RNA, DNA, non-coding small or large RNAs
(i.e. siRNA, miRNA, shRNA), DNA, mRNA- or shRNA-expression
plasmids, DNA, protein, ligand, receptor, cytokine, stimulating or
deactivating antibody, pharmacological agent, feeder cells.
7. A cell modification device according to claim 1, wherein the
cell modifying surface is located on the inner surface of the
centrifugation chamber, a spiral-shaped element or on at least one
cylindrical element.
8. A cell modification device according to claim 1, characterized
in that the centrifugation chamber comprises at least two cell
modifying surfaces which are functionalized with the same or
different at least one substance enhancing proliferation of cells,
and/or inducing genetic modification and/or inducing cellular
modification of cells.
9. A method for modifying cells comprising the steps introducing
cells in a cell modification device, comprising a centrifugation
chamber with at least one cell modifying surface with a normal
vector having an angle of 135-45.degree. to the rotational axis of
the centrifugation chamber wherein and comprising at least one
input/output port, immobilizing the cells on the cell modifying
surfaces by the rotation of the centrifugation chamber at 2 to 2000
g maintaining the rotation of the rotation of the centrifugation
chamber until the cells are modified.
10. A method according to claim 9 wherein the cells are modified by
bringing into contact with at least one cell modifying surface
functionalized with at least one substance enhancing proliferation
of cells, and/or inducing genetic modification and/or inducing
cellular modification of cells.
11. A method according to claim 9 wherein the cells are modified by
bringing into contact with at least one cell modifying surface
functionalized with particles being functionalized with at least
one substance enhancing proliferation of cells, and/or inducing
genetic modification and/or inducing cellular modification of
cells.
12. A method according to claim 9 wherein the cells are modified by
immobilizing cells on at least one cell modifying surface and
bringing into contact with particles being functionalized with at
least one substance enhancing proliferation of cells, and/or
inducing genetic modification and/or inducing cellular modification
of cells.
13. A method according to claim 9 wherein the cells are subjected
to at least two different gravitational forces.
14. A cell composition modified by a method according to claim
1.
15. A cell composition of claim 14, wherein the cell composition
has at least two layers, the layers comprising modified cells of
different cell types or cells of a different phenotype.
16. A system for cell modification, comprising: a centrifugation
chamber with at least one cell modifying surface with a normal
vector having an angle of 135-45.degree. to the rotational axis of
the centrifugation chamber and at least one input/output port a
device to rotate the centrifugation chamber so as to apply a
centrifugal force to cells.
Description
DISCLOSURE OF INVENTION
[0001] The present invention relates to methods and devices for
modifying eukaryotic cells on functionalized surfaces of a
centrifugation apparatus
BACKGROUND OF THE INVENTION
[0002] The conditions during cell culturing have a substantial
impact on the phenotypes of the cells and desired or not, cell
culturing leads to the manipulation of cells.
[0003] Cell culture refers to methods under which eukaryotic cells,
especially of mammalian origin, are maintained at appropriate
conditions with supply of cell culture medium in a cell incubator
or a fermenter. Cell culture conditions vary widely depending on
the cell type and the desired application. Variation of cell
culture conditions can be utilized for cell expansion, cell
differentiation or manufacturing of different phenotypes of the
cell type. The most commonly varied factor in culture systems is
the cell culture medium, for which a vast number of recipes is
known (see for example "Cell Culture Techniques" Humana Press, 1st.
Edition, 2011).
[0004] Typically culture systems utilize a large amount of medium
compared to the mass of the cells to provide a sufficient reservoir
for nutrients. In static systems, the medium covering the cells is
limiting the gas diffusion to the cells if the cell culture surface
itself does not allow gas diffusion. Slow macroscopic convection of
the medium results in uncontrolled and uneven supply of nutrients
to the cells and may result in different differentiated i.e.
manipulated cells.
[0005] Culturing large numbers of cells adhered to a surface
without the use of carriers or large volume cell suspension is
difficult and requires frequent change of the medium. The known
static systems for cell culturing are labor-intensive and need
clean room conditions during handling the cell cultures, for
example media exchange or transfer cells from and into storing
devices or adequate incubators for proper cell growth. In dynamic
systems for cell culturing like roller fermenters, cells can
dislocate from the surface of the fermenter and are suspended in
the media. The conditions for growing and supply of nutrients is
not uniform for adhered and suspended cells and will result in
different differentiated or modified cells. Centrifugation systems
for the separation or modification of cells are known.
[0006] It is long known to separate cells from a cell mixture into
fractions of different cell types with the aid of centrifugal
forces in a centrifuge according to their density i.e. their
sedimentation velocity. The cell separation is carried out in a
specially designed centrifuge, rotor and container (flask) for the
cells. For example, whole blood is fractionated or separated by
centrifugation into blood plasma (as upper phase), buffy coat (thin
layer of leukocytes mixed with platelets in the middle phase), and
erythrocytes as lower phase.
[0007] The effect of enhanced gravity generated by centrifugation
on cells under culturing conditions has been investigated in
various publications. Huang et al (2009) disclose in "Gravity, a
regulation factor in the differentiation of rat bone marrow
mesenchymal stem cells" in J. Biomed. Here, rBMSCs are first plated
on glass coverslips; after 24 h the cells had adhered to the
coverslips and the coverslips were transferred to a biocompatible
polyethylene culture bag, are incubated with medium and then
cultured on a cell centrifuge at 2 g hypergravity for several days.
The medium was changed every 3 days during HG/SMG culture.
[0008] Gaubin et al. described in Microgravity Sci. Technol. 1991
February; 3(4):246-50 the effects of hypergravity on adherent human
cells. Galimberti et al disclose in "Hypergravity speeds up the
development of T-lymphocyte motility", Eur Biophys J, May 1, 2006;
35(5): 393-400 a hypergravity cell culture for 1 to 11 days. Cell
culture is performed in flasks which were positioned vertically to
the centrifugation axis in the centrifuge. The use of flasks within
a centrifuge is furthermore proposed by Versati et al in "Effects
of gravity on proliferation and differentiation of adipose
tissue-derived stem cells", J Gravit Physiol, 14(1): P127-128
(2007). Here, a commercial available medium sized centrifuge
(MidiCAR) is used to accommodate cell culture flasks to investigate
cell growth under hypergravity conditions. Morbidelli et al.
investigated in Microgravity Sci. technol (2009) 21:135-140 the
effect of hypergravity on endothelial cell function and gene
expression. Cell manipulation or cell modification is not disclosed
in this publication.
[0009] The methods disclosed in these publications are with the
exception of hypergravity conditions nearly identical to common
cell culturing and involve manual handling steps like medium
change. Change of medium i.e. the supply of cells to be cultured
with nutrients involves stopping of the centrifugation process,
thereby interruption of the enhanced gravitational forces. Manual
handling steps are not only laborious and prone to contamination,
but also destroy the micro environment of the cells like cell/cell
contact or cell/cell interaction. An unaffected micro environment
of the cells is important for cell cultivation, e.g. for the
activation of lymphocytes or viral or retroviral transduction
processes. There is no disclosure in the prior art about the nature
of the surface of the flasks or the centrifugation chamber.
[0010] It is further known that retroviral transduction of cells
can be accelerated by hypergravity, for example described by Tonks
et al in Biotechnol Prog. 2005; 21(3): 953-8. With this technique,
retrovirus vectors are coated on plates and cells are brought into
contact with the virus. In order to promote the contact between
target cells and the virus vector, the plate comprising adhered
virus and cells are placed into a centrifuge. This requires manual
handling steps and the cells are not supplied with medium during
centrifugation.
[0011] WO 2009/072003 discloses a centrifugation system for cell
proliferation. Cell manipulation or cell modification is not
disclosed in this publication.
[0012] The invention provides a novel device and method for
modifying cell populations on functionalized cell modifying
surfaces under hypergravity conditions generated by the rotation of
a centrifugation chamber. With the device and method of the
invention, eukaryotic cells can be modified and/or eukaryotic cells
with new or modified features can be generated.
SUMMARY OF THE INVENTION
[0013] It is a first object of the invention to provide a cell
modification device, comprising a centrifugation chamber with at
least one cell modifying surface with a normal vector having an
angle of 135-45.degree. to the rotational axis of the
centrifugation chamber, wherein the centrifugation chamber
comprises at least one input/output port and the cells to be
modified are immobilized at the cell modifying surfaces by the
rotation of the centrifugation chamber at 2 to 2000 g.
[0014] The device of the invention comprises a centrifugation
chamber with at least one input/output port through which cells,
cell culturing liquids (media), gases and other materials can enter
and leave the chamber without the need of stopping the rotation of
the centrifugation chamber. The device comprises preferable one
input port and one output port for liquids and at least one,
especially two for gases.
[0015] Another object of the invention is a method for modifying
cells comprising the steps [0016] introducing cells in a cell
modification device, comprising a centrifugation chamber with at
least one cell modifying surface with a normal vector having an
angle of 135-45.degree. to the rotational axis of the
centrifugation chamber wherein and comprising at least one
input/output port, [0017] immobilizing the cells on the cell
modifying surfaces by the rotation of the centrifugation chamber at
2 to 2000 g [0018] maintaining the rotation of the rotation of the
centrifugation chamber until the cells are modified.
[0019] Cell modification according to the invention relates to all
methods where cells are kept physiologically active and are
modified. The modification may result for example in a change of
the phenotype, function, number or differentiation status of the
cells, like
[0020] a) cell division, differentiation or cell proliferation
[0021] b) activation of a signal transduction cascade
[0022] c) change of the cellular activation status and/or cell
function
[0023] d) genetic modification of cells
[0024] e) growing of layers of different or identical cell types
involving cell-cell contact
[0025] The modification of the cells results for example in a
change of expression of certain proteins, of RNA molecules, of
miRNA, in a change of post translational modification, in a change
of DNA methylation or in histone modification.
[0026] The cell modification device comprises cell modifying
surfaces which can be functionalized for cell modification.
[0027] The mechanical/chemical stimulus changing the phenotypes of
the cells is provided or triggered by the functionalized cell
modifying surfaces of the centrifugation chamber of the invention.
The term "functionalized surface" as used in this application
includes all types of surfaces which can provide a stimulus to a
cell. Typically, functionalized cell modifying surface comprise a
coating of chemical or physical immobilized bioactive compounds,
like [0028] proteins, peptides, nucleic acids; [0029] spacer
molecules enhancing the adhesion of cells or bioactive compounds to
the cell modifying surfaces like hydrophilic polymers
(functionalized poly lactate, polyvinyl alcohols, polysaccharides;
functionalized dextranes); [0030] organic or inorganic particles as
carrier of bioactive compounds, especially magnetic particles
coated with functionalized poly lactate, polyvinyl alcohols or
functionalized dextranes; [0031] substances enhancing cell
adhesion, e.g polypeptides, lipids, polysaccharides; [0032] viruses
and retroviruses or particles thereof [0033] cells which can be
used for modification of a target cell, such as antigen presenting
cells, "accessory cells" producing certain bioactive factors or
cell lines transfected with certain functional molecules. [0034]
stimulus provided by mitogens, cytokines, stimulatory antibodies or
receptor ligands
[0035] The cell modification device according to the invention
comprises at least one cell modifying surface which is
functionalized for example for adherence, proliferation, genetic
and/or cellular modification of the cells, or for proliferation of
cells in one or more layers.
[0036] The cell modification device according to the invention
comprises preferable at least one cell modifying surface which is
functionalized with at least one substance enhancing proliferation
of cells, and/or inducing genetic modification and/or inducing
cellular modification of cells. The cell modifying surface can
further be functionalized with particles being functionalized with
at least one substance enhancing proliferation of cells, and/or
inducing genetic modification and/or inducing cellular modification
of cells.
Surface Functionalization with Cell Binding Systems
[0037] In a first embodiment of the invention, the cell modifying
surfaces may be functionalized with any substance which is suitable
for cell culture and useful or required to introduce preferable
cell culture conditions for a given cell type.
[0038] The cell modifying surfaces can be functionalized in order
to enhance adherence and/or proliferation of cells on the cell
modifying surfaces. Suitable substances for functionalization of
the surfaces are glycoproteins, polypeptides, glycosaminoglycans,
disccharides, biotin binding molecules or protein tags. For
example, the surface may be coated with extracellular matrix
proteins including all collagen types (I to VIII).
[0039] Furthermore, the cell modifying surfaces may comprise an
affinity binding system. One of the most widely used affinity
binding system is the avidin-biotin or streptavidin-biotin system.
For example, the cell modifying surface may be first coated with
avidin and/or streptavidin (or derivates thereof) to facilitate
binding of a biotinylated molecule like a biotinylated antibody. It
is furthermore possible to coat the cell modifying surface first
with biotin (or derivates thereof) to facilitate binding of another
molecule functionalized with streptavidin and/or avidin. Both
variants result in high affinity binding of the second molecule to
the cell modifying surfaces. The strong interaction between
streptavidin or avidin-biotin is made much weaker by using a
combination of modified streptavidin or avidin and modified biotin
like desthiobiotin or a derivative thereof like DSB-X Biotin
(Hirsch et al. 2002: "Easily reversible desthiobiotin binding to
streptavidin, avidin, and other biotin-binding proteins: uses for
protein labeling, detection, and isolation". Analytical
Biochemistry 308: 343-357; US2008/0255004A1). A protein, such as an
antibody may be biotinylated with the modified biotin. When this
protein is immobilized by binding the modified biotin to an
optionally modified streptavidin or avidin molecule bound to the
cell modifying surface, it may be released under mild conditions by
adding free biotin.
[0040] The functionalizing of the cell modifying surface like
coating with biotin or (strept)avidin may be performed before or
during the process of the invention, both inside or outside of the
centrifugation chamber or the device of the invention. The renewal
of the coating or the functionalization of the cell modifying
surface may be performed between two process steps and without
interruption of the rotation of the centrifugation chamber. For
example, the renewal of the functionalized cell modifying surface
is possible by adding biotinylated molecules or molecules with
(strept)avidin to a cell modifying surface which is coated with
streptavidin or biotin, respectively.
[0041] Further affinity binding systems suitable for the cell
modifying surfaces comprise antibodies, for example antibodies
against biotin or protein tags for example IIsopeptago, BCCP or
Myc-tag.
[0042] The cell modifying surfaces may be further be coated with
libraries of substances synthesized with methods of combinatorial
chemistry in order to identify substances which work best as
binding system for a given cell type.
[0043] Certain bioactive polymers may be used as spacer molecules
enhancing the adhesion of cells or the binding of other substances
on the cell modifying surfaces like functionalized poly lactic
acid, polyvinyl alcohols, polysaccharides or dextranes or derivates
thereof. This binding system is especially useful as basic coating
of a cell modifying surface produced from a hydrophobic plastic
material like poly carbonate, polystyrene or polyethylene. The cell
modifying surfaces may be coated with highly reactive polymers as
e.g. disclosed in U.S. Pat. No. 6,977,138B2.
[0044] The cell modifying surfaces can comprise one or more
substances which enhance adhesion and/or proliferation of cells.
Especially useful are one or more substances selected from the
group consisting of collagen types (I to VIII), fibronectin,
gelatin, laminin, elastin, hyaluronic acid, keratan sulfate,
chondroitin sulfate, heparan sulfate proteoglycans, poly-d-lysine,
avidin, streptavidin, biotin, antibodies, antibodies against biotin
or protein tags, protein tags like IIsopeptag, BCCP, Myc-tag,
Calmodulin-tag, FLAG-tag, HA-tag, His-tag, Maltose binding
protein-tag, Nus-tag, Glutathione-S-transferase-tag, Green
fluorescent protein-tag, Thioredoxin-tag, S-tag, Softag 1, Softag
3, Strep-tag, SBP-tag, Ty tag, certia, poly lactate, polyvinyl
alcohols, polysaccharides and dextran.
Surface Functionalization for Cellular Modification
[0045] In a second embodiment of the method of invention, cell
modification comprises cellular modification like activation,
proliferation, dedifferentiation and/or differentiation of cells.
Accordingly, the cell modifying surfaces may be functionalized with
any substance which is suitable for cellular modification of cells
like cell activation, proliferation, dedifferentiation and
differentiation of cells. The cell modifying surface can further be
functionalized with particles being functionalized with at least
one substance suitable for cellular modification of cells like cell
activation, proliferation, dedifferentiation and differentiation of
cells.
[0046] Particular, cell modification by the method and the device
of the invention comprises the alteration of gene expression,
protein expression, post-translational or posttranscriptional
modifications of genes, mRNAs or proteins, protein phosphorylation,
histone modification, or modification of intracellular signaling
cascades (e.g. Ca2+ influx).
[0047] Furthermore, cellular modification may comprise cell
activation for example by agonistic or antagonistic antibodies,
cytokines, growth factors, (de-)activating ligands,
pharmacologically active substances, mitogens, DNA or RNA-modifying
substances.
[0048] The cell modifying surfaces can be functionalized for one or
more cellular modification steps.
Surface Functionalization for Genetic Modification
[0049] In a third embodiment of the invention, the cell modifying
surfaces may be functionalized with any substance which is suitable
for genetic modification of cells, i.e. modification of cells using
genetic material or any other substances interacting, binding or
integrating into cellular polynucleotides or the genome and/or
altering their function. Again, the cell modifying surface can
further be functionalized with particles being functionalized with
at least one substance suitable for genetic modification of cells,
i.e. modification of cells using genetic material or any other
substances interacting, binding or integrating into cellular
polynucleotides or the genome and/or altering their function.
[0050] Genectic modification of a cell according to this invention
includes for example transduction by viral, such as adeno-viral or
retroviral or lentiviral vectors or transfection with nucleic
acids, i.e. coding RNAs, non-coding small or large RNAs (i.e.
siRNA, miRNA, shRNA), DNA, mRNA- or shRNA-expression plasmids or
other substances interacting or binding or integrating into
cellular polynucleotides or the genome and/or altering their
function.
[0051] Genetic modification furthermore comprises contacting the
cells for example with a virus, viral particle, RNA, DNA, protein,
ligand, receptor, cytokine, stimulating or deactivating antibody,
pharmacological agent, other cells (e.g. feeder cells) or layers of
several cells or cell types. The contacting agent can be soluble in
the cell culturing liquid or attached to the cell modifying
surfaces, or can be expressed or anchored to the surface of another
cell used for co-culture.
[0052] The cell modifying surfaces can be functionalized for one or
more genetic modification steps.
Surface Functionalization for Cell Layers
[0053] Culturing cells on flat cell modifying surfaces often
results in two-dimensional sheets, which is an artificial
environment for any cell. Eukaryotic cells experience in vivo a
three-dimensional environment and are surrounded by other cells,
membranes, fibrous layers and adhesion proteins. Three-dimensional
cell cultures are known and use as support extracellular matrices,
scaffolds and proteins to provide an in vivo-like morphology and
physiologically relevant environment. Commercially available 3D
cell culture systems are e.g. MaxGel.TM. human Extracellular Matrix
(ECM), HydroMatrix.TM. synthetic peptide, and mouse ECM, from
Sigma.RTM. to support stem cell and other cell cultures.
[0054] A forth object of the invention is to provide a layered cell
composition, wherein cells are grown in a layered system like
tissue or organs. For this purpose, the device and the method of
the invention is used to immobilize cells at defined positions,
e.g. in successive layers of same or different cell types, and to
keep the cells at a fixed position by the centrifugal forces,
allowing building of complex layers. In addition to be grown in a
layered system, the cells may further be modified as described
above.
[0055] The cell modifying surfaces of the device of the invention
can comprise one or a plurality of identical or different
functionalized cell modifying surfaces. For example, the cell
modifying surfaces can be equipped with an affinity binding system
in addition to functionalization of the surface for genetic
modification of the cells.
BRIEF DESCRIPTION OF DRAWINGS
[0056] FIG. 1 is a schematic view of a cell modification device
used in embodiments of the invention.
[0057] FIG. 2 shows an embodiment of the invention with a conical
shaped chamber having culturing surfaces with a normal vector
sharing an angle different than 90.degree. (for example
105.degree.) with the rotational axis (g).
[0058] FIG. 3 shows another embodiment of the device of the
invention, wherein the chamber and/or the element have a conical
bottom or base plate (b) and at least one aperture or tube (h)
reaching to the bottom of the chamber and/or the element.
[0059] FIG. 4 shows several embodiments of centrifugation chambers
with a plurality of internal structures or concentric elements in
top view.
[0060] FIG. 5 shows an embodiment with two cell modifying surfaces;
the first cell modifying surface (b) having a normal vector of
about 90.degree. to the rotational axis of the centrifugation
chamber and the second cell modifying surface (e) having a normal
vector of about 0.degree. to the rotational axis of the
centrifugation chamber.
[0061] FIGS. 6 and 7 show a variant with concentric or
spiral-shaped cell modifying surfaces (e) with a normal vector
having an angle of 135-45.degree. (shown 90.degree.) to the
rotational axis of the centrifugation chamber and a second cell
modifying surface (f) with a normal vector having an angle of
(-45)-45.degree. (shown with an angle of 0.degree.) to the
rotational axis of the centrifugation chamber.
[0062] FIG. 8 shows an embodiment in which the cell modifying
surfaces (e) are not or not throughout connected to the second cell
modifying surface f) and the top cover of the chamber, thereby
allowing a flow of cell culturing liquid and gases via tubing or
channels c' and d'. Optionally tubing or channel d' comprises
apertures for distribution of the cell culturing liquid and gases
over the cell modifying surfaces (e).
[0063] FIG. 9 shows an embodiment in which concentric or
spiral-shaped cell modifying surfaces (f) with a normal vector
having an angle of 135-45.degree. (shown 90.degree.) to the
rotational axis of the centrifugation chamber and second cell
modifying surface (h) with a normal vector having an angle of
(-45)-45.degree. (shown 0.degree.) to the rotational axis of the
centrifugation chamber are combined.
DETAILED DESCRIPTION OF THE INVENTION
[0064] In general, cell modification of the invention involves cell
culturing conditions where cells are kept physiological active over
a period of time. This is usually accomplished at temperatures of
25-45.degree. C. and with a supply of nutrients like glucose and
gases like O.sub.2 and CO.sub.2. During the culturing process, the
conditions can be maintained stable or are subject to changes such
as hyper/hypoxia conditions, in-/decreased pressure, different
gravitational forces, in-/decreased supply of nutrients or growth
factors, in-/decreased temperature, high or low cell density,
in-/decreased medium osmolarity, or gradients of nutrients,
chemokines/cytokines/growth factors or stimulatory/deactivating
antibodies.
Cell Media
[0065] In the method of the invention, various cell culturing
liquid (media) known in the art of cell culturing can be used as
stimulus for cells, including one or more of the following media
DMEM, HBSS, DPBS, RPMI, Iscove's medium, X-VIVO.TM., each
optionally supplemented e.g. with fetal calf serum, human serum or
serum substitutes or other nutrients or cell stimuli like
Cytokines. The media can be standard cell media like the above
mentioned media or special media for e.g. primary human cell
culture (e.g. for endothelia cells, hepatocytes or keratinocytes)
or stem cells (e.g. dendritic cell maturation, hematopoietic
expansion, keratonocytes, mesenchymal stem cells or T cell
expansion). The media may have supplements or reagents well known
in the art, e.g. albumins and transport proteins, amino acids and
vitamins, antibiotics, attachments factors, growth factors and
cytokines, hormones or solubilising agents. Various media are
commercially available e. g. from LifeTechnologies or
Sigma-Aldrich.
Centrifugation Conditions
[0066] During cell modification in the device and the method of the
invention, the cells to be modified are immobilized at the cell
modifying surfaces by the gravitational forces due to the rotation
of the centrifugation chamber.
[0067] The invention is preferably carried out at a rotational
speed of the centrifugation chamber generating centrifugal forces
of more than 1 g and up to 2000 g, preferable between 20 and 1000
g, more preferable between 20 and 500 g and especially preferable
between 20 and 100 g.
[0068] The degree of cell modification can be adjusted by the speed
of rotation of the centrifugation chamber, since the gravitational
forces enacting on the cells depend on the speed of the
centrifugation chamber, density of the culturing media, density of
the cells and the distance of an individual cell to the rotational
axis of the centrifugation chamber.
[0069] The magnitude of centrifugal forces F acting on a given cell
depends on the mass m of the cell, its speed, i.e. its angular
velocity w, and the radius r of curvature, i.e. the distance
between the cell and the rotational axis of the chamber, according
to the following formula
F=mr.omega..sup.2
[0070] The mass m of the cell is calculated from the cell volume
(V.sub.cell) and the cell density (.delta..sub.c0). Cell density
.delta..sub.cell of eukaryotic cells is between 1.04 and 1.09
g/cm.sup.3. Taking into account the buoyant force relative to the
media density (.delta..sub.media), the centrifugal force F can be
calculated as follows
F=(.delta..sub.cell-.delta..sub.media)V.sub.cellr.omega..sup.2
[0071] The angular velocity can be expressed as rotations of the
chamber per time (2.pi./T). If a individual cell is located at the
inner wall of the chamber, r equals the inner radius of the
chamber.
[0072] The degree of interaction between surface and cell may be
modified changing the density of the medium. Typically, media
density (.delta..sub.media) is around 1.0 g/cm.sup.3, but can be
changed by appropriated additives. Accordingly, cells can be
released during the process of the invention from the cell
modifying surfaces by utilizing a cell medium with a higher density
or enhancing the density of the cell medium by adding appropriated
additives.
[0073] The cell modification according to the invention involves
centrifugation conditions applied to the cells as long as necessary
to induce the desired modification of the cells. The duration of
the centrifugal forces depends on the desired modification of the
cells and is not limited. Centrifugal forces may be applied to the
cells during the process of the invention for as short as 10 s or
as long as 10 days. Typically, centrifugal forces of more than 2 g,
especially more than 5 g or more than 10 g are applied for at least
40, 120 or 360 minutes up to 720 minutes.
[0074] It is also possible to maintain centrifugation at the same
speed during the entire process or to use a sequence of several
(2-50) periods of centrifugal forces with same or different speed
of rotation. The duration of the centrifugal forces may vary,
depending on the desired modification of the cells. For example,
the speed of rotation may be higher if a process step for genetic
and/or cellular modifications of cells is involved compared to
rotational speed during steps for culturing and/or expanding the
cells. The continuous flow of liquid through the centrifugation
chamber and/or over the cell modifying surfaces can be achieved
through variation of the centrifugal forces i.e. through a
variation of speeds of rotation of the chamber.
Use of Particles
[0075] Modification of cells with the device and method of the
invention may further comprise the use of particles, especially
particles having functionalized (i.e. biologically active)
surfaces. The particles may be produced from organic material like
polymers (poly dextranes, poly saccarides, poly styrene, poly
lactides or polyvinyl alcohol, each chemically modified or
unmodified) or inorganic material like silica, alumina or
ferromagnetic metals or metal oxides. Particles made from inorganic
material may be coated with the polymers mentioned. The size of the
particles depends on their intended function and may vary between
20 nm and 500 .mu.m.
[0076] Preferable, the particles are coated or at least doped with
biologically active substances. The biologically active substances
may be mixed with the bulk material of the particle and can be
released during the process of the invention. In another variant,
the biologically active substances are only present on the outer
surface of the particles.
[0077] The particles may contain or be coated with all biologically
active substances already disclosed in the present application for
surface functionalization for cell layers, surface
functionalization with cell binding systems, surface
functionalization for cellular modification or surface
functionalization for genetic modification.
[0078] Particles may be coated or immobilized by the centrifugal
forces on the cell modifying surfaces before introducing the cells
to be modified into the centrifugation chamber. In this case, the
cells are immobilized by the centrifugal forces on the particles.
In another variant or the method of the invention, first the cells
to be modified are immobilized by the centrifugal forces on the
cell modifying surfaces. Then, the particles are introduced into
the centrifugation chamber, for example as suspension in the cell
media. In this variant of the invention, the particles are
immobilized by the centrifugal forces on the cells.
[0079] The particles and/or biologically active substances are
brought into close contact with the cells to be modified with the
aid of the centrifugal forces exerted on the cell membrane of the
cells. Depending on the centrifugal forces exerted on the cell
membrane of the cells, it is even possible that the particles
and/or biologically active substances are introduced into the
cells. Substances which transiently permeabilize the cell membrane
can be added to assist this process.
[0080] Particles can be used in any process step of the invention,
alone or in addition to other disclosed biologically active
substances or coatings.
Sequence of Processing Steps
[0081] In another embodiment of the method of the invention, the
cells are subjected to a sequence of at least two different
gravitational forces i.e. rotational speeds of the centrifugation
chamber. In this embodiment, at least two different process steps
can be performed, each with a rotational speed adapted for the
respective process step.
[0082] A sequence of same or different centrifugal forces applied
on the cells (i.e. rotational speed of the centrifugation chamber)
allows the control of the kind or the degree of cell modification.
For example, the cells can be genetically modified by transducing
with virus particles in a first processing step at a rotational
speed generating centrifugal forces of 100 g to 1000 g and
thereafter cultured/expanded in a second processing step at a
rotational speed generating centrifugal forces of 2 g to 100 g.
[0083] The method of the invention can comprise a sequence of
processing steps consisting of at least two centrifugation steps
with the same or different centrifugal forces applied which are
optionally interrupted by for example the change or renewal of the
cell modifying surfaces or culturing media, or the addition of
stimulating substances or cells. The exchange or renewal of any
material can be performed during the process of the invention
without opening the centrifugation chamber.
[0084] For example, the method of the invention can comprise a
sequence of processing steps, wherein cells are first introduced
into the chamber and immobilized at the functionalized cultural
surfaces by the rotation of the centrifugation chamber. After a
first modification, like a proliferation step, the cells are rinsed
at low rotational speed of the chamber from the cell modifying
surfaces into a buffer container via the inlet/outlet port. Then,
the centrifugation chamber may be stopped and a new (same or
different) coating may be applied to the cell modifying surfaces.
In an alternative variant of the invention, the rotation of the
chamber is not stopped, and the cell modifying surfaces are coated
with the same (fresh) or a different functionalized coating under
ongoing rotation of the chamber. An affinity binding system as
disclosed above may be used for a recoating step.
[0085] After the cell modifying surfaces are replaced or recoated,
the cells are reintroduced from the buffer container into the
centrifugation chamber and the next modification step under
centrifugation conditions can be performed.
[0086] In a further example for a sequence of processing steps
during the process of the invention, the cell modifying surface may
first be coated e.g. with BD Primaria.TM. to enhance the
proliferation of the cells and then with virus particles for one or
more transduction steps. The cell modifying surface may be recoated
with new (same or different) virus particles between two
transduction processes. For functionalizing the cell modifying
surface with virus particles, the cells are rinsed from the
surfaces and stored in a buffer container. After the coating
process, the cells are reintroduced into the centrifugation chamber
and the second culturing step can be started.
Batch and Continuous Modification
[0087] The centrifugation chamber and the method of the invention
permit both the batch-wise and the continuous modification of
cells. In a batch-wise modification, the cells either stay during
the whole process within the chamber or are completely removed and
after an intermediate step reintroduced into the chamber. Batch
processing involves usually an intermediate storage of cells in a
buffer container.
[0088] Continuous modification means that the cells are
continuously introduced into and removed from the chamber during
the modification process. Continuous modification involves e.g. a
conical shaped centrifugation chamber or cell modifying surfaces
and/or a flow of media through the chamber which transports cells
as required. For continuous modification, the centrifugation
chamber comprises at least two inlet/outlet ports for liquids and
gases and optionally an intermediate storage of cells in a buffer
container.
[0089] Introducing the cells in the chamber, rinsing cells into a
buffer container, washing and coating of the cell modifying
surfaces and reintroducing the cells into the chamber can be
performed with the aid of pumps and tubes and controlled e.g. by
appropriate software.
Supply of Nutrients and Overall Conditions
[0090] Temperature and gas composition of the centrifugation
chamber can be controlled and adjusted if appropriate for the cell
types or the modification steps to be performed. For this purpose,
a heating and/or cooling means can be attached to the device of the
invention.
[0091] In the method of the invention, it is preferred to cover the
cells to be modified with a layer of liquid (media) as thin as
possible to supply the cells with gases such as O.sub.2, N.sub.2
and CO.sub.2 by diffusion. The thinner the film, the easier
diffusion of gases and the better cells can be supplied. Therefore
in another variant of the invention, the cell culturing liquid is
moved over or relative to the cells e.g. by changes of the
rotational speed or by adding additional media through the ports.
Preferable, the liquid media is moved over the cells during
rotation of the chamber in form of a liquid film with a thickness
of less than 50 .mu.m, less than 100 .mu.m, less than 200 .mu.m,
less than 500 .mu.m, less than 1000 .mu.m or less than 2000 .mu.m.
Films of cell culturing liquids having such thickness are
sufficient to cover and supply the cells with the necessary
nutrients and gases. The cells may be supplied with cell culturing
liquids by constant movement of the liquid relative to the
cells.
[0092] In another variant of the invention, the cell culturing
liquids are exchanged or renewed during the modification process in
a constant flow. For this variant, the device of the invention has
at least two ports for inlet/outlet of cell culturing liquid. The
exchange of liquids can be performed without stopping the rotation
of the centrifugation chamber.
[0093] The cell culturing liquid (media) supplied to the cells may
have the same composition during the entire modification process.
It is furthermore possible to change the composition of the media
during the modification process, for example by withdrawing a first
medium and supplying a second medium from/to the chamber or by a
constant flow of medium with a constant change of composition.
Cells to be Modified
[0094] The eukaryotic cells modified in the device and/or the
method according to the invention may origin from any mammalian or
human source, such as tumor, blood, tissue, bone marrow or cell
lines, for example one or more cell types selected from the group
consisting of human cells, fibroblasts, embryonic stem cells,
keratinocytes, melanocytes, mesenchymal stem cells, epithelial
cells, T-cells, regulatory T-cells, B-cells, NK-cells, neuronal
cells, dendritic cells, stem cells (adult, embryonic, hemapoietic),
cells originating from epithelium, ectoderm, endoderm, endothelium,
mesoderm, epithelial tissue, basal lamina, vasculature, Connective
tissue, fibrous tissues, Muscle tissue, visceral or smooth muscle,
skeletal muscle, cardiac muscle, nervous tissue, brain, spinal
cord, cranial nerves, spinal nerves or motor neurons.
[0095] The method and the device of the invention are especially
suitable for modification of eucaryotic cells, preferable for
modification of one or more cell types selected from the group of
human blood and immune system cells consisting of Megakaryocyte
(platelet precursor), Monocyte, Connective tissue macrophage
(various types), Epidermal Langerhans cell, Osteoclast (in bone),
Dendritic cell; lymphoid tissues), Microglial cell (in central
nervous system), Neutrophil granulocyte, Eosinophil granulocyte,
Basophil granulocyte, Mast cell, Helper T cell, Suppressor T cell,
Cytotoxic T cell, Natural Killer T cell, B cell, Natural killer
cell, Reticulocyte, Stem cells and committed progenitors for the
blood and immune system (various types), and tissue or tumor stem
cells.
[0096] According to method of the invention at least two different
cell types or cells of at least two different phenotypes can be
modified.
[0097] The cells exhibit a different phenotype after modification.
It is a further object of the invention to provide a cell
composition modified by the method of the invention. Yet another
object of the invention is to provide a cell composition with at
least two layers, the layers comprising modified cells of different
cell types or cells of a different phenotype.
Modification Techniques
[0098] It is an advantage of the cell culturing device and method
according to the invention that the cells are pressed against the
cell modifying surfaces by the centrifugal forces, thereby
enlarging the cell surface adjacent to the functionalized cell
modifying surfaces. Enlarging the cell surface enhances the chances
of contact between for example a target cell to be modified and a
feeder cell or a retrovirus.
[0099] Furthermore, the centrifugal forces bring the functionalized
cultural surfaces in close contact with the membrane of the cells
to be modified. The close contact causes the cell to act for
example by signal transduction or uptake of the extracellular
material into the cell. Modification techniques during the method
of the invention may comprise genetic or cellular modification of
the cells or the preparation of cellular layers.
Genetic Modification
[0100] The term "genetic modification of cells" refers to all
processes manipulating the genetic program of a cell on the level
of DNA, RNA or translation of RNA into proteins by introduction of
oligo- and/or polynucleotides into the genetic material of the
cell. The transfected material may be only transiently expressed,
e.g. in form of plasmids within the cell, or the transfected
material may be stably expressed by integration of the genetic
material into the genome of the cell. Genetic modification during
the method of the invention comprises all techniques of molecular
cloning and transformation to alter the structure and
characteristics of the genes of a cell to be modified. This may
include using recombinant nucleic acid (DNA or RNA) techniques to
form new combinations of heritable genetic material followed by the
incorporation of such material into the cell.
[0101] The process of the invention may comprise various methods of
introducing foreign nucleic acids into a eukaryotic cell, which are
known to the skilled artisan.
[0102] Such methods include applying physical treatment, like, for
example, applying nanoparticles or magnetofection, using chemical
materials like cyclodextrin or cationic polymers such as
DEAE-dextran or polyethylenimine or using biological particles
(viruses) that are used as carriers.
[0103] Genetic modification of cells within the method of the
invention comprises furthermore the use of genetic modifying agents
resulting in a genetic modification of the cell. Such genetic
modifying agents are nucleic acids, e.g. DNA or RNA. The nucleic
acid may be naked or in complexes with carrier molecules such as
polymers, liposomes, or microparticles. The DNA may be in linear
form (oligonucleotides, polynucleotides) or in circularized form
(e.g. DNA-plasmids). The RNA may be any kind of RNA known to exist
in the cell (e.g. mRNA, miRNA, siRNA, shRNA). The nucleic acid (DNA
or RNA) may be derivatives of the naturally occurring nucleic acids
or may be chemically modified. For example, modified nucleotides
may include: linked nuclear acid (LNA), 2-O-Me nucleotides,
2'-O-methoxyethyl, and T fluoro. Backbone modifications include,
for example, phosphorothioate and phosphate.
[0104] Another genetic modifying agent are viral-based gene
delivery system which involves genetically engineered recombinant
viruses, like, for example, Adenovirus, Adeno-Associated Virus,
Retrovirus, Vaccinia virus and Lentivirus, which carry the gene of
interest in their capsid.
[0105] Genetic modifying agent also comprises chemical mutagens
such as base analogues (e.g. 5-bromouracil (5-BU)) which are
incorporated into DNA, agents modifying purines and pyridines or
agents labilizing bases (e.g. nitrous oxide, hydroxylamine and
alkylating agents) and agents producing distortions in DNA (e.g.
fluorescent acridine dyes such as proflavine and acridine
orange.
[0106] Genetic modification of cells within the method of the
invention comprises for example introducing the nucleic acids, e.g.
DNA or RNA, into the cell by using the already disclosed particles.
The nucleic acid to be introduced into the cell may be covalently
or non-covalently attached to the surface of the particles
resulting in nucleic acid particle complexes. The nucleic acid
particle complex may be immobilized on the cell modifying surface
of the centrifugation chamber or the nucleic acid particle
complexes may be given into the liquid/media within the
centrifugation chamber. Then application of gravitational forces by
rotation of the centrifugation chamber of the present invention
drives the nucleic acid particle complexes towards and into the
target cells, where the cargo is released.
[0107] Genetic modification of cells within the method of the
invention comprises for example introducing the nucleic acids, e.g.
DNA or RNA, into the cell using chemical-based transfection agents
such as e.g. cyclodextrin, polymers, liposomes. The complexes of
nucleic acid, e.g. DNA (linear or in circular form, e.g. plasmid)
or RNA, and the chemical transfection agents, e.g.
Lipofectamin.RTM. may be immobilized on the cell modifying surface
of the centrifugation chamber. Then application of gravitational
forces by rotation of the centrifugation chamber of the present
invention drives the complexes of nucleic acid, e.g. DNA or RNA,
and the chemical transfection agents towards and into the target
cells. Alternatively, the complexes of nucleic acid, e.g. DNA or
RNA, and the chemical transfection agents, e.g. Lipofectamin.RTM.
may be given into the liquid/media within the centrifugation
chamber resulting in transfection of the cell during the
centrifugation of the centrifugation chamber.
[0108] Genetic modification of cells within the method of the
invention comprises for example introducing the nucleic acids, e.g.
DNA or RNA, into the cell using viral-based gene delivery systems
(e.g. adenovirus, adeno-associated virus, retrovirus, and
lentivirus). The virus or virus particles to be introduced into the
cell may be covalently or non-covalently attached to the surface of
the cell modifying surface of the centrifugation chamber or the
virus or virus particles may be given into the liquid/media of the
centrifugation chamber. Then application of gravitational forces by
rotation of the centrifugation chamber of the present invention
drives the virus or virus particles towards and into the target
cells.
[0109] In some embodiments of the invention, cell modifying
surfaces are optionally coated with affinity binding systems i.e.
peptides enhancing retroviral transduction like for example,
RetroNectin.RTM. (Takara, Japan). The multivalent nature of such
affinity binding systems allows the simultaneous binding of cells
and viruses, bringing the two into close physical proximity. The
co-localization of viruses and cells facilitates infection,
resulting in higher frequencies of stable gene transfer. Affinity
binding systems may furthermore be coated on particles, which
results in a co-localization of viruses and cells on the particles.
The particles itself may be coated on the cell modification surface
or may be utilized in suspension and immobilized on the cells by
centrifugation.
[0110] In other embodiments of the invention the cell modifying
surfaces are functionalized with modified, e.g. pseudotyped,
viruses as vectors such as disclosed in WO2008/037458. Vectors
derived from the gamma-retroviruses, for example, the murine
leukemia virus (MLV), have become a standard tool for gene transfer
technology and have been frequently used in clinical gene therapy
trials (Ross et al., Hum. Gen Ther. 7:1781-1790, 1996).
Pseudotyping of retroviral vectors, including HIV vectors or MLV
vectors, refers to the incorporation of envelope proteins from
heterologous viruses into the retroviral envelope membrane. Such
pseudotyped retroviral vectors then exhibit a receptor phenotype
similar to the virus from which the envelope protein was derived.
Depending on the host range of said virus, the pseudotyped
retroviral vectors will then have a broadened or a narrowed host
range as compared to vector particles having the incorporated
homologous retroviral envelope proteins. Useful pseudotyped vectors
include MLV vectors pseudotyped with the HIV Env protein, the Ebola
virus glycoprotein, or the baculovirus glycoprotein.
[0111] The measles virus (MeV), a prototype morbillivirus of the
genus Paramyxoviridae, utilizes two envelope glycoproteins (the
fusion protein (F) and the hemagglutinin protein (H)) to gain entry
into the target cell. WO2008/037458 discloses the pseudotyping of
retroviral vectors with heterologous envelope proteins derived from
the Paramyxoviridae family, genus Morbillivirus. The incorporation
of morbillivirus F and H proteins having truncated cytoplasmic
tails into lentiviral vector particles allows an effective
transduction of cells. In addition, these pseudotyped vector
particles allow the targeted gene transfer into a given cell type
of interest by modifying a mutated and truncated H protein with a
single-chain antibody or ligand directed against a cell surface
marker of the target cell, e.g. the stem cell marker CD133.
Cellular Modification
[0112] The term "modification of cells" refers to all processes
which result in a morphological, functional, or molecular
modification of the cells (e.g. activation, proliferation,
reprogramming, dedifferentiation, differentiation or maturation).
This embodiment of the invention comprises techniques like cell
activation or stimulation for example by agonistic or antagonistic
antibodies or cytokines or the in vitro modulation of cells like
the in vitro expansion and/or genetic modification of lymphocytes.
For example, T-lymphocytes can be cultured with antibodies against
cell surface molecules like CD3 either bound to a macroscopic
matrix like the cell modifying surfaces of the invention or in
soluble form in the presence of antigen presenting cells, e.g.
using peripheral blood mononuclear cells (PBMC) or fractions
thereof as feeder cells and polyclonal stimuli. Instead of CD3
antibodies, specific antigens can be used for the stimulation and
expansion of antigen-specific T-cell. In these types of cultures
viral transductions of the T cells or any other type of genetic
modification as described above can also be performed as already
described, to achieve cellular modifications.
[0113] The cellular modification of cells within the method of the
invention comprises for example the use of feeder cells or
modifying cells that secrete certain metabolites, growth or
differentiating factors into the medium or that directly deliver
signals to the cells to be modified.
[0114] Feeder cultures, which secrete growth factors, can be
prepared from splenocytes, macrophages, thymocytes, or fibroblasts.
E.g. mouse embryonic fibroblasts (MEFs) are often used as feeder
cells in human embryonic stem cell research. Genetically modified
cells, such as K562 cells, stably transfected with stimulatory
molecules, e.g. MHC class I or MHC class II, ligands for
costimulatory molecules CD28, ICOS, Notch, CD137, CD40 or
cytokines, e.g. IL-2 or IL-15 or facilitating molecules, e.g.
Fc-gamma receptor (for labelling with Fc-bearing stimulatory
molecules, e.g. antibodies or Fc-fusion proteins) can also be
used.
[0115] The cellular modification with the method of the invention
may further comprise the delivery of transcription factors (TFs)
into cells promoting differentiation, transdifferentiation or
dedifferentiation/reprogramming of the target cells. In this
embodiment of the invention, the method comprises altering the
state of a cell, for example an adult somatic cell, embryonic or
adult stem cell, or a mesenchymal stem cell (MSC) by introducing
one or more transcription factors or substances, which alter the
expression or activity of said transcription factors, into the
cells. The cells then alter the expression level of at least one
polypeptide (e.g. Oct3/4 for an induced pluripotent stem cell)
and/or epigenetic programming of the cell is changed.
[0116] Introducing the transcription factor into the target cells
can be achieved by contacting a cell with a transcription factor, a
polypeptide or fragment thereof fused to a protein transduction
domain which allows entry of the protein into the cell or by any
other means to transport active substances as defined above into
cells and thereby altering the expression profile and/or epigenetic
status, e.g. leading to reprogramming the cells. For example, Xie
et al (2004, Cell: 117:663-676) disclose a method for the forced
expression of a single TF to trigger a specialized B cell to
transdifferentiate into a macrophage.
[0117] Cellular modification of cells during the methods of the
invention can be further achieved with a cocktail of extrinsic
signaling molecules to enhance differentiation and widen the
spectrum of MSC plasticity. A suitable method to deliver TFs into
MSCs is disclosed by Brazilay et al (2009), Stem Cells,
27:2509-2515.
[0118] The method of the invention is especially suited for the
modification and expansion of T cells, either polyclonal or
antigen-specific. The interaction of T-cells with a stimulatory
agent like a stimulatory antibody or specific MHC/peptide complex
on the surface of an antigen presenting cells (APC) can be
increased by the increased gravitational force during
centrifugation. For this purpose, the cultural surfaces can be
coated with T cell stimulatory molecules like stimulatory
antibodies against CD3, CD28 or CD137. It is advantageously to
activate the T-cells to be modified with stimulatory antibodies in
a soluble form or with particles coated with stimulatory antibodies
during the process of the invention. Furthermore, T-cells can be
co-cultured with APC, like T-cell depleted PBMC or artificial APC
(e.g. K562 cells, transfected with Fc-gamma receptor and/or MHC
molecules and/or costimulatory molecules, like CD137 ligand, or
CD28 ligands), in various ratios (e.g.: 10:1 to 1:1000 T
cells/APC).
[0119] Instead of stimulatory antibodies, T-cells can be
co-cultured with specific antigens, e.g. defined antigenic
peptides, purified defined proteins or protein mixtures or lysates
of defined pathogens. This type of culture could be useful for
activation or expansion of antigen-specific T-cells. Furthermore,
any kind of T cell stimulatory agent can be used within the method
of the invention, e.g. PMA, ionomycin, superantigens like SEB,
lectins, like ConA or PHA.
[0120] The method of the invention allows the regulation of the
interaction of T-cells with stimulating substances or cells via the
centrifugation time and/or rotational speed. The interaction
between the cells to be modified, the cultural surfaces and the
substances or cells (like APC) applied to the centrifugation
chamber can repeated as required to restimulate the cells or
initiate their expansion. Furthermore, fresh media, cytokines other
substances relevant for the cell modification/culture can be added
in an automated fashion, without the necessity to interrupt the
interaction between cells and coated surface or APC.
[0121] The above mentioned substances, ligands, factors, agents,
particles or cells may be applied, coated or adhered to the
cultural surfaces or introduced into the centrifugation chamber
with the culturing liquids.
Cellular Layers
[0122] A layered cell composition according to the invention
comprises at least two layers of cells with the same or different
cell type or phenotype. Preferable, the layered cell composition
comprises 2 to 10, especially 2 to 5 layers of cells with different
cell type or phenotype. Each of these layers may comprise one or
more (like 10 to 50) layers of the same cell type. Layered
compositions of the invention may consist of complex cellular
tissue, like stem cells on top of feeder cells, skin tissue or
organs and may comprise same or different types of cells for
example stem cells, fibroblasts, keratinocytes, melanocytes,
ephitelial cells, endothelial cells, antigen-presenting cells (B
cells, dendritic cells, macrophages).
[0123] In this embodiment of the invention, for example cells of a
first type are cultured on the cell modifying surface of the
centrifugation chamber. On this first layer, cells of a second type
are placed or immobilized by the centrifugal forces, which
furthermore enhances the contact and interaction between the cells
of the first and second type. Further layers or cell types can be
placed on the existing cell layers resulting in a multilayer cell
structure. In addition, matrices can be used for culturing the
cells in three-dimensional structures. Such matrices are for
example three-dimensional lattices e.g. proteoglycans, collagen or
artificial matrices useful for culturing cells in three
dimensions.
[0124] With the method and devices of the invention, it is possible
to generate layered cell composition resembling human skin. Such
layered cell compositions may be used, for example, as artificial
skin.
Devices According to the Invention
[0125] A schematic view of a cell modification device according to
the invention is shown in FIG. 1 with centrifugation chamber (a),
rotational axis (g) and culturing surfaces (e). The culturing
surfaces can be positioned parallel to the rotational axis (g),
i.e. the normal vector of the culturing surfaces shares an angle of
90.degree. with the rotational axis (g). By rotation of the chamber
by axis (g), cells (f) are immobilized at the culturing surfaces
(e) and can be supplied with cell culturing medium via at least one
inlet/outlet port, like the shown inlet (c) and outlet port
(d).
[0126] Devices of the invention may be equipped with one port which
is used for both the introduction and removal of cells, media or
gases into or out of the chamber. In another variant, at least two
ports, for example one inlet and one outlet port for liquids and
one or more ports for gas exchange are used. The ports are
preferably integrated into the rotational axis of the
centrifugation chamber and may in case of one inlet and one outlet
port be attached from the same or from different sides of the
centrifugation chamber.
[0127] A conical shaped chamber having culturing surfaces with a
normal vector sharing an angle different than 90.degree. (for
example 105.degree.) with the rotational axis (g) is shown in FIG.
2. In this embodiment, the cells and the media can move over the
cell modifying surface depending on the rotational speed towards
the side of the chamber having the wider diameter (in FIG. 2:
upward). This can be advantageously used for genetic modification
of the cells, for example with a cell modifying surface coated with
virus particles for retroviral transduction. By movement of the
cells over the surface, the contact area of the cells to the
surface is enhanced, thereby enhancing the chance for cell
modification like retroviral transduction. Furthermore, the cells
are supplied by the movement of media over the cells in form of a
thin film.
[0128] If the method of the invention comprises at a processing
step wherein the cells are moving (or forced) over the cell
modifying surface during rotation of the chamber, it is preferable
to employ at least two different rotational speeds of the
centrifugation chamber. For example in a first processing step, a
higher rotational speed resulting in centrifugal forces of 100 g to
1000 g moves the cells towards the side of the chamber having the
wider diameter and in a second processing step at lower rotational
speed or even stopped chamber the cells slide down the cell
modifying surfaces towards the base plate b). The processing steps
of at least two different rotational speeds may be repeated as
often as needed to achieve the desired level of cell
modification.
[0129] FIG. 3 shows another embodiment of the device of the
invention, wherein the chamber and/or the element have a conical
bottom or base plate (b) and at least one aperture or tube (h)
reaching to the bottom of the chamber and/or the element. During
rotation, the cells (f) are immobilized at the cultural surfaces
(e). If the rotation of the chamber is too slow or even stopped,
the cells will accumulate at the lowest point (i) of the conical
bottom or base plate (b) and can be removed by the internal tube
(h) and outlet port (d).
[0130] The centrifugation chamber comprises at least one cell
modifying surface at which the cells are immobilized by the
rotation of the centrifugation chamber. The cell modifying surface
is located in the centrifugation chamber or on the inner surface of
the centrifugation chamber and may have any three dimensional shape
like a wall or barrier as thin as mechanically possible with a
height according to the sample size or the cell population to be
modified.
[0131] The cell modifying surface may be located on the inner
surface of the centrifugation chamber, a spiral-shaped element or
on at least one cylindrical element.
[0132] The cell modifying surface may be located on at least one
cylindrical element or structure like a wall or a layer. The number
of cylindrical elements depends on the volume of the centrifugation
chamber and/or the number of cells to be modified/cultured. In
alternative, the cell modifying surface may be in shape of a spiral
with or without an opening to the outside of the spiral to avoid
the loss of medium due to centrifugal forces.
[0133] In another embodiment of the invention, the cell modifying
surfaces are located on or are a part of an element insertable into
the centrifugation chamber. Preferably, the cell modifying surfaces
and/or the cylindrical element and the structures therein may
comprise apertures or segments to facilitate the flow of medium to
any part of the cell modifying surfaces in order to supply all
cells immobilized on the cell modifying surface in sufficient
manner. The cell modifying surfaces, the cylindrical element or the
internal structures may furthermore comprise an appropriate number
of spacer elements to ensure the mechanical stability of the cell
modifying surfaces during centrifugation and to ensure the free
flowing of cell culture liquid and gases through the chamber.
[0134] Cell modifying surfaces in form of a spiral can be obtained
by winding up a film or foil to form a coil. Cell modifying
surfaces located on a coiled film can be used without apertures or
segments, since the liquid is forced through the chamber by the
centrifugal forces. In another variant, the film comprises spacer
elements to ease the flow of liquids between the film layers. The
coil of film can be inserted in the chamber or into an appropriate
concentric element to form a spiral. By using a film as substrate
for the cell modifying surfaces, high surface areas for high cell
densities or cell numbers can be provided.
[0135] FIG. 4 shows several embodiments of centrifugation chambers
with a plurality of internal structures or concentric elements in
top view. Label (193) denominates the rotational axis and (194) the
outer wall of the chamber. The cell modifying surfaces are labelled
with (191) and (192) and may be concentric or spiral-shaped
elements. The cell modifying surfaces can comprise spacer elements
(195) generating sufficient space between the cell modifying
surfaces for free flowing of cell culture liquid and gases.
[0136] It is furthermore possible that the centrifugation chamber
comprises at least two cell modifying surfaces which are
functionalized with the same or different at least one substance
enhancing proliferation of cells, and/or inducing genetic
modification and/or inducing cellular modification of cells. The
cell modifying surfaces may have different functionality or
different coated surfaces. In this embodiment, the device may
comprise at least a first cell modifying surface with a normal
vector having an angle of 135-45.degree. to the rotational axis of
the centrifugation chamber and at least a second cell modifying
surface with a normal vector having an angle of (-45)-45.degree. to
the rotational axis of the centrifugation chamber.
[0137] For example, the cell modifying surfaces with a normal
vector having an angle of 135-45.degree. to the rotational axis of
the centrifugation chamber can be functionalized for genetic
modification of the cells, whereas the cell modifying surfaces with
a normal vector having an angle of (-45)-45.degree. to the
rotational axis of the centrifugation chamber can be functionalized
for proliferation of the cells. FIG. 5 shows this embodiment, with
the first cell modifying surface (b) having a normal vector of
about 90.degree. to the rotational axis of the centrifugation
chamber and the second cell modifying surface (e) having a normal
vector of about 0.degree. to the rotational axis of the
centrifugation chamber. This embodiment of the invention allows at
least two different modification steps at two different cell
modifying surfaces in one chamber without the need to change the
cell modifying surfaces during the process.
[0138] FIGS. 6 and 7 show another variant of this embodiment by way
of example with concentric or spiral-shaped cell modifying surfaces
(e) with a normal vector having an angle of 135-45.degree. (shown
90.degree.) to the rotational axis of the centrifugation chamber
and a second cell modifying surface (f) with a normal vector having
an angle of (-45)-45.degree. (shown with an angle of 0.degree.) to
the rotational axis of the centrifugation chamber. The
centrifugation chamber shown in FIG. 6 is in centrifugation state,
where all cells are immobilized at the cell modifying surfaces (e)
by the centrifugal forces. FIG. 7 shows the device after stopping
the rotation of the chamber around axis b, the cells are rinsed
from the cell modifying surfaces (e) and can be further cultured on
the cell modifying surface (f) as shown in FIG. 7.
[0139] The cell culturing liquid may be supplied in a constant flow
or is moved by variations of the speed of rotation over the cells.
For example, in FIG. 8, the cell modifying surfaces (e) are not or
not throughout connected to the second cell modifying surface f)
and the top cover of the chamber, thereby allowing a flow of cell
culturing liquid and gases via tubing or channels c' and d'.
Optionally tubing or channel d' comprises apertures for
distribution of the cell culturing liquid and gases over the cell
modifying surfaces (e).
[0140] The chamber may comprise at least one aperture allowing a
flow of cell culturing liquid and/or gases into and out of the
chamber. The aperture is preferable located in the axis (g) of the
centrifugal chamber or concentric element as shown in FIG. 8. The
cell culturing liquid and/or gases are supplied via inlet and
outlet port c/d located in the rotational axis (g) and are then
forced by the centrifugal movement over the cultural surfaces. The
cell culturing liquids can be either withdrawn from the system via
tubing or channel d' or directed back into the moulded element or
the centrifugal chamber via bypass (c').
[0141] FIG. 9 shows another embodiment of the invention, in which
concentric or spiral-shaped cell modifying surfaces (f) with a
normal vector having an angle of 135-45.degree. (shown 90.degree.)
to the rotational axis of the centrifugation chamber and second
cell modifying surface (h) with a normal vector having an angle of
(-45)-45.degree. (shown 0.degree.) to the rotational axis of the
centrifugation chamber are combined. In this embodiment, the second
cell modifying surfaces are attached to the first cultural surface
(f) in a way that cells may be easily be transferred from the first
to the second cultural surface and vice versa by change of
rotational speed of the chamber. In this embodiment, the first and
second cultural surfaces have a different functionalized coating
thereby providing different modification to the cells.
[0142] The concentric elements as supporting structures for the
cultural surfaces, the cultural surfaces itself and/or the
centrifugation chamber may be made of various materials, preferably
from plastics like, for example, polystyrene (PS),
polyvinylchloride (PVC), polycarbonate, glass, poly acrylate, poly
acrylamide, polymethylmethacrylate (PMMA), polyethylene
terephthalate (PET), poly tetrafluorethylen (PTFE), thermoplastic
polyurethane (TPU), silicone, poly ethylene (PE) poly propylene
(PP), polyvinyl alcohol (PVA) or compositions comprising one or
more of the above mentioned materials. In a preferred embodiment,
the cell modifying surfaces may be coated with a biodegradable
material, for example, collagen, chitin, alginate, and/or
hyaluronic acid derivatives, poly lactic acid (PLA) polyglycolic
acid (PGA) and their copolymers.
[0143] The size of the centrifugation chamber depends on the number
of cells to be modified and may have the size of 2 cm to 50 cm in
diameter and a height of 5 mm to 50 cm.
[0144] The centrifugation chamber of the device of the invention
may be a single component with the cultural surfaces and/or
supporting structures like concentric elements for the cultural
surfaces. In another embodiment of the invention, the
centrifugation chamber consists of an outer chamber (for example
made from stainless steel) in which one or more concentric elements
made from the above mentioned materials can be inserted. The cell
modifying surfaces are then located on or are a part of the
concentric elements.
[0145] The concentric elements may be disposable (i.e. single use)
or may be designed and manufactured for re-use after washing and
sterilization.
[0146] Furthermore, the cell modifying surfaces can be
rough-textured, grooved and/or may comprise pockets or recesses to
enhance the adherence of the cells to be cultured.
[0147] The process of the invention can be automated for example in
a sample processing system as known from EP 0869838B1 and WO
2009/072003. The methods described here allow for automation in a
closed cell modification device eliminating the risk of
contamination of the cell culture compared to a standard non-closed
transduction process, especially when the transduction process is
repeated several-fold. In addition, safety of the operator is
increased due to reduction of direct contact with biological
hazardous material like retroviruses.
Systems According to the Invention
[0148] Yet another object of the invention are systems for cell
modification, comprising:
[0149] a) a centrifugation chamber with at least one cell modifying
surface with a normal vector having an angle of 135-45.degree. to
the rotational axis of the centrifugation chamber and at least one
input/output port
[0150] b) a device to rotate the centrifugation chamber so as to
apply a centrifugal force to cells
[0151] The systems may furthermore comprise
[0152] c) at least one container containing the cells to be
modified
[0153] d) at least one container for the cells to be modified
[0154] e) at least one container containing cell media
[0155] f) a tubing set connecting centrifugation chamber and
container
[0156] g) at least one pump and
[0157] h) a plurality of valves
[0158] The systems for cell modification can be operated by
controlling the device to rotate the centrifugation chamber, the
pump and the valves to introduce the cells to be modified and cell
media into the centrifugation chamber, rotate the centrifugation
chamber and remove modified cells from the centrifugation
chamber.
[0159] The system of the present invention can include various
mechanical, electromechanical, and magnetic components. A system
according to the invention is shown in FIG. 10, wherein the
centrifugation chamber 128 having input/output port 130 can be
connected to pump 108 and a plurality of valves 110. Container for
the cells to be modified, the modified target cells and cell media
are not shown but can be placed on hooks 114.
[0160] The system can optionally include a magnetic separation unit
106 with housing for positioning a separation column like a
magnetic separation column.
[0161] The system 100 further includes a pump 108 and a plurality
of fluid flow control means or valves, as illustrated by one or
more valves 110. The components of the system 100 (e.g.,
centrifugation chamber, valves, pump, separation unit, etc.) can be
coupled or connected by one or more flow paths so as to form a
series of fluid pathways or fluid circuits. The system further
includes a computer control system or unit 112 providing monitoring
and/or control of one or more aspects of the system 100. The
computer system 112, as described above, can include one or more
input and/or output devices, graphical displays, user interfaces
and may allow for manual and/or automated control of system 100
operation and functions. The computer control system 112 can
include a module or system to process information (e.g., flow
information, etc.) within the system 100 and can include a wide
variety of proprietary and/or commercially available computers,
components or electronics having one or more processing structures
and the like, with such systems often comprising data processing
hardware and/or software configured to implement any one or a
combination of method steps as described herein. Software will
typically comprise machine readable code of programming
instructions embodied in a tangible media such as a memory, digital
or optical recording media, optical, electrical, or wireless
telemetry signals, or the like, and one or more of these structures
may also be used to output or transmit data, signals, or
information between components of the system in any of a wide
variety of signal processing architectures.
[0162] The system can further include various supports, sensors,
housings, etc. for various components that can be coupled with the
present system to perform methods as described herein.
[0163] The system 100 further include one or more support
structures 114 configured to hold and/or support various fluids,
reagents, samples fluid reservoirs, filters, and the like that can
be utilized with the system 100 according to the present invention.
Support structures cart include various hook or hanger, or holder
(e.g., filter holder or housing) configurations and are not limited
to any particular design. Fluids, buffers, reagents, etc.
positioned on a support 114 can be coupled to a fluid path or
tubing, that can in turn be connected to more or more components of
the system 100. The system 100 can include sensors for monitoring
and/or further controlling fluid flow through the system. Sensors
can include, for example, liquid sensors, which can include bubble
detectors (ultrasonic detector), pressure sensors, and the like.
Bubble detector 116 and pressure sensors 118 are shown. A support
120 is show, which can be configured to hold a filter or volume
reduction unit. Collection area 122 can support collection
containers, reagents, etc.
[0164] Processing unit 104 can include a housing or cover 124, that
can be movable (e.g., removable) about one or more hinge. The cover
124 at least partially defines a processing area 126 that can be
temperature controlled and coupled to temperature monitoring and
control components that may be housed within the housing 105 of the
system 100. The processing unit 104 includes a centrifugation
chamber 128 configured for holding and processing (e.g.,
centrifugation, culturing, sample component separation, etc.) of a
sample. The centrifugation chamber 128 shown is a rotating chamber
held in position about an axis that can include an anti-rotation
lock 130. The processing unit 104 can include one or more detection
systems, such as an optical detector 132 positioned within the
cover 124 and configured to detect or monitor processing of a
sample in the chamber 128. One or more fluid input/output lines can
be coupled to the chamber 128 and may be held in position by a
holder 134.
[0165] All publications referred to herein are hereby incorporated
by reference in their entirety.
Examples
[0166] The Examples described below are to exemplify the apparatus,
methods, and systems of the invention and are not intended to limit
the disclosure of the invention as described herein.
Example 1
Viral Transduction of T Cells with Disease-Specific T Cell Receptor
Genes
[0167] A use of the invention is the introduction of genes coding
for a disease-specific T cell receptor into a polyclonal population
of T cells, which may then be used for therapeutic injection into
patients. The T cells are directed towards the target antigen, e.g.
tumor cell or infected cells.
[0168] A centrifugation chamber providing cell modifying surfaces
coated with RetroNectin.RTM. is supplied with a recombinant virus
containing supernatant, wherein the virus encodes the target
antigen, and rotated at surfaces by the gravitational forces
generated by the rotation. Following this coating step, the chamber
is rotated at low rotation speed and the T cells to be modified are
introduced into the high rotational speed (e.g. 2000.times.g) for 2
hours. For improved viral transduction, the T cells are previously
activated, e.g. by cultivation in the presence of antibodies
against CD3 and CD28, either in the same centrifugation chamber or
in a separate device. By centrifugation (e.g. 1000.times.g for 15
min) the T cells in the chamber come into intimate contact with the
virus coated surface, allowing viral transduction. The
centrifugation speed is adjusted to optimize the transduction.
Transient lowering of the centrifugation speed allows detachment of
the cells and subsequent centrifugation at high speed reattaches
the cells at another point of the coated surface. This process can
be repeated several times, e.g. to achieve multiple interactions of
the cells with virus coated surfaces. Following this transduction
process the rotation speed is stopped or reduced to a minimum, i.e.
sufficient to keep the cells at the cultivation surface. During the
process optimal cell culture media, containing appropriate amounts
of nutrients and growth factors is added continuously to the
chamber via the inlet port of the rotary chamber system. The
centrifugation fixes the cells at a certain location, and therefore
media can be added and removed without changing the location of the
cell, i.e. without interfering with the modification process. The
constant exchange of the medium without affecting the cell
position, i.e. modification process, also allows to use a minimal
medium volume at a given time, i.e. the distance of the cell
attached to the culture surface to the gas reservoir/medium surface
can be <5 mm. In this way optimal gas supply is guaranteed
without the need for a steady state large medium volume, usually
used as a reservoir of nutrients.
[0169] During the transduction process of high speed and/or lower
speed, a steady flow of stimulation media over the cells or cell
culture via the inlet and outlet port of the chamber is maintained.
This removes transduction inhibitors and improves the target cell
viability.
[0170] Each transduction process is adjusted to the optimal
interaction of the cells with the virus particles (depending on
cell and virus type) coated to the surface of the centrifugation
chamber or moulded element by adaptation of the centrifugation
speed (increasing or reducing the g number) leading efficient,
fast, easy and safe handling of the transduction process.
Example 2
Activation and Expansion of Antigen-Specific T Cells
[0171] T cells can be activated and expanded by antigens loaded in
or on antigen-presenting cells (APC). T cell activation requires
intimate contact between the T cells and APC.
[0172] To improve T cell activation a system described herein is
used to spin down APC and T cells in an appropriate ratio, e.g.
1:100 to 100:1. Either physiological cell mixtures such as PBMC,
containing T cell and APC or defined cell preparations, e.g.
purified T cells and APC, e.g. dendritic cells, B cells,
macrophages, cell lines transfected with distinct MHC molecules,
etc, mixed at an appropriate ratio are used. In addition antigens,
proteins, peptides, cell lysates, and growth factors and/or
co-stimulatory antibodies, e.g. anti CD28, antiCD137, may be added.
The contact between the cells is rapidly induced and maintained at
an appropriate level by centrifugation.
[0173] APC and T cells can be deposited in distinct layers, e.g. T
cell on top of a layer of APC, enabling optimal contact of T cells
to APC. In conventional culturing devices, cells slowly sediment in
an uncontrolled fashion providing asynchronous and only suboptimal
contact between APC and T cells. During cultivation centrifugation
fixes the cells at a distinct position and therefore media, growth
factors, co stimulatory molecules or antigens can be added in a
controlled fashion without disturbing the cellular interaction. By
changing the centrifugational speed the interaction between the
cells is modulated at different phases of the culturing process,
e.g. inducing firm contact at an early phase and reduced contact at
later phases. This results in an accelerated and synchronous and
more pronounced activation of T cells and in addition allows
optimal control of the cellular microenvironment in terms of
cellular composition, supply with nutrients, growth factors etc.
Under these conditions the rapid and controlled activation of
antigen-specific T cells is achieved.
[0174] The activated T cells are further purified, e.g. based on
the expression of activation markers, such as cytokines, CD154 or
CD137 by magnetic cell separation. Such cells can be generated
against various antigens, e.g. pathogens, tumors or, in case of
regulatory T cells against auto antigens. These cells can be used
for cellular therapies.
[0175] A particular advantage of the invention is the fact, that
the whole cell cultivation process including all described
manipulations required to achieve optimal results can be performed
in a closed system, i.e. with minimal risk of contaminations.
Example 3
Polyclonal Activation and Expansion of T Cells
[0176] The systems of the invention provide an optimized platform
for polyclonal activation and expansion of T cells, comprising
conventional T cells or regulatory T cells.
[0177] This example is similar to Example 2 except that instead of
defined antigen, polyclonal stimuli are used, comprising antibodies
against CD3 and co-stimulatory molecules, such as CD28 and/or
CD137. These antibodies are added either in soluble form, requiring
the addition of accessory cells bearing Fc-receptors, e.g.
conventional antigen-presenting cells or cell lines transfected
with Fc-receptors. Alternatively the added antibodies are
immobilised on a macroscopic surface, e.g. a particle or bead
ranging from about 30 nm to 100 .mu.m. These immobilised antibodies
are directly cultured with purified T cells, e.g. at ratios 1:4 to
4:1. As described above the system used allows regulated contact of
T cells and stimulating agent and controlled addition of additional
environmental factors, e.g. nutrients, cytokines, etc.
[0178] The polyclonal populations of T cells generated can be used
in cellular therapies, e.g. polyclonal regulatory T cells for
treatment of autoimmune or graft versus host disease or the
prevention of organ transplantation.
* * * * *