U.S. patent application number 17/538403 was filed with the patent office on 2022-03-17 for cell growth matrix.
The applicant listed for this patent is UNIVERCELLS TECHNOLOGIES S.A.. Invention is credited to Jose Castillo, Bastien Mairesse, Quentin Vanwalleghem.
Application Number | 20220081664 17/538403 |
Document ID | / |
Family ID | 1000006000337 |
Filed Date | 2022-03-17 |
United States Patent
Application |
20220081664 |
Kind Code |
A1 |
Castillo; Jose ; et
al. |
March 17, 2022 |
CELL GROWTH MATRIX
Abstract
The invention provides a structured cell growth matrix or
assembly comprising a one or more spacer layers and one or more
cell immobilization layers. The invention further provides a
bioreactor comprising said matrix or assembly.
Inventors: |
Castillo; Jose; (Brussels,
BE) ; Mairesse; Bastien; (Uccle, BE) ;
Vanwalleghem; Quentin; (Uccle, BE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERCELLS TECHNOLOGIES S.A. |
Nivelles |
|
BE |
|
|
Family ID: |
1000006000337 |
Appl. No.: |
17/538403 |
Filed: |
November 30, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16933398 |
Jul 20, 2020 |
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17538403 |
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15938800 |
Mar 28, 2018 |
10876090 |
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16933398 |
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PCT/EP2017/078775 |
Nov 9, 2017 |
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15938800 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 23/22 20130101;
C12M 25/02 20130101; C12M 23/20 20130101; C12M 25/14 20130101; C12M
25/06 20130101; C12M 1/18 20130101; C12M 41/46 20130101; C12M 23/44
20130101; C12M 23/06 20130101; C12M 23/28 20130101; C12N 5/0068
20130101; C12N 2533/90 20130101; C12M 25/16 20130101; C12N 2535/00
20130101; C12N 5/06 20130101 |
International
Class: |
C12M 1/12 20060101
C12M001/12; C12M 3/00 20060101 C12M003/00; C12N 5/00 20060101
C12N005/00; C12M 1/00 20060101 C12M001/00; C12M 1/18 20060101
C12M001/18; C12M 1/34 20060101 C12M001/34; C12N 5/07 20060101
C12N005/07 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 9, 2016 |
BE |
BE2016/5839 |
Claims
1. An apparatus for culturing cells, comprising: a bioreactor
including a matrix assembly for growing cells in connection with a
cell medium flowing through the matrix assembly, the matrix
assembly comprising at least two cell immobilization layers in
direct contact.
2. The apparatus of claim 1, the bioreactor having a fluid flow
direction aligned with a major dimension of the matrix
assembly.
3. The apparatus of claim 1, wherein the at least two cell
immobilization layers comprise woven layers.
4. The apparatus of claim 1, wherein the at least two cell
immobilization layers comprise woven mesh layers.
5. The apparatus of claim 1, further including at least one
non-woven layer.
6. An apparatus for culturing cells, comprising: a bioreactor
including a matrix assembly for growing cells in connection with a
cell medium flowing through the matrix assembly, the matrix
assembly comprising at least one woven mesh layer and at least one
non-woven layer.
7. The apparatus of claim 6, wherein the matrix assembly comprises
a plurality of woven mesh layers.
8. The apparatus of claim 7, wherein the plurality of woven mesh
layers comprise at least a first woven mesh layer in direct contact
with at least a second woven mesh layer.
9. The apparatus of claim 6, wherein the at least one non-woven
layer is in contact with one of the first or second woven mesh
layers.
10. An apparatus for culturing cells, comprising: a bioreactor
including a matrix assembly for growing cells in connection with a
cell medium flowing through the matrix assembly, the matrix
assembly comprising at least one woven mesh layer.
11. The apparatus of claim 10, wherein the matrix assembly
comprises a plurality of woven mesh layers.
12. The apparatus of claim 11, wherein the plurality of woven mesh
layers comprise at least two woven mesh layers in direct
contact.
13. The apparatus of claim 12, wherein the plurality of woven mesh
layers are arranged in a stacked configuration.
14. The apparatus of claim 12, further including a plurality of
cell immobilization layers arranged in a stacked configuration with
the plurality of woven mesh layers.
15. The apparatus of claim 10, further including a plurality of
cell immobilization layers each comprising a nonwoven material.
16. The apparatus of claim 10, wherein the bioreactor comprises a
roller bottle.
17. The apparatus of claim 10, wherein the bioreactor is adapted
for passing the cell medium in a vertical flow through the matrix
assembly.
18. The apparatus of claim 17, wherein the bioreactor is adapted to
create the vertical flow through the matrix assembly from bottom to
top.
19. The apparatus of claim 17, wherein the bioreactor is adapted to
create the vertical flow through the matrix assembly from top to
bottom.
20. The apparatus of claim 10, wherein the matrix assembly is
spiral wound.
21. The apparatus of claim 10, wherein the matrix assembly is
spiral wound about a core.
22. The apparatus of claim 10, wherein the matrix assembly is
annular.
23. The apparatus of claim 10, wherein the at least one woven mesh
layer comprises openings having a shape selected from the group
consisting of round, elliptical, square, or rectangular.
24. The apparatus of claim 10, wherein the at least one woven mesh
layer comprises openings having a size of between at least 0.05 mm
and 5 mm.
25. The apparatus of claim 10, the bioreactor having a fluid flow
direction along the at least one woven mesh layer.
26. The apparatus of claim 10, further including at least one
non-woven layer.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 16/933,398 filed on Jul. 20, 2020, which is a continuation of
U.S. application Ser. No. 15/938,800 filed on Mar. 28, 2018, which
is a continuation of PCT/EP2017/078775 filed on Nov. 9, 2017, which
claims priority to Belgium application 13E2016/5839 filed on Nov.
9, 2016, the disclosures of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention concerns cell growth matrix, in
particular a structured, high density cell growth matrix. The
invention further concerns the use of the matrix for growing cells
and a bioreactor comprising said matrix.
BACKGROUND
[0003] The techniques to culture cells such as eukaryotic cells,
animal cells, mammalian cells and/or tissue are difficult and
complex since these cells are delicate and have nutrient and oxygen
requirements during growth which are complex and difficult to
maintain. Given the increasing need to culture cells in large
quantities, bioreactors and culturing devices have become an
important tool in research and in the production of cells for
producing active proteins and/or antibodies and/or any cell
by-products.
[0004] The bioreactors of the prior art comprise non-structured
cell growth matrixes and are provided with internal or external
circulation mechanisms for cell culture medium circulation. The
cell growth matrixes generally comprise carriers which can have the
form of beads with regular or irregular structure, or may comprise
woven or non-woven microfibers of a polymer or any other material
compatible with cell growth. The carriers can have a variety of
forms and dimensions.
[0005] Several drawbacks can be attributed to the known bioreactors
and their non-structured cell growth matrixes. The reproducibility
lack of volumetric homogeneity of growth matrixes during their
packing, and their movement and redistribution over time under the
influence of the culture medium flow can lead to unreproducible
cell culture environment and very different micro-environments in
different portions of the bioreactor. The carriers might gather in
a given area inside the bioreactor thereby considerably lowering
homogeneity during cell culture. Consequently, part of the cultured
cells will have very limited or no access to the culture medium
and/or oxygen supply, which results in different cells metabolism,
differences in product quality, even the death of said cells and a
low cells production rate. Additionally, the non-homogenous
matrixes are difficult to produce and to pack given the variable
sizes and shapes of the carriers which might stick to each other.
Another drawback of the cell growth matrixes of the prior art is
that their cell culture surface cannot be determined with
precision. Lastly, packing the carriers in the bioreactor is
laborious and thus costly.
[0006] U.S. Pat. No. 3,948,732 describes an assembly which includes
a spiral-wound tubular chamber unit through which culture media and
cells travel within the chamber and about the center of the spiral
and adhere to the inside wall of the chamber. The assembly includes
a spacer member provided with spaced projecting support members
which is interleaved with the tubular chamber unit. These
projecting members are intended to facilitate gas flow through the
cross section of the spiral assembly in an axial direction. At no
time does the spacer member come into contact with any of the cells
or cell culture medium within the tube much less provide a path for
the cells in any way.
[0007] It is the aim of the present invention to provide a cell
growth matrix and a bioreactor comprising said matrix which
overcome at least part of the above mentioned drawbacks. By
preference, the cell growth matrix should provide for a large cell
growth surface within a small volume while still allowing
circulation of medium and cells. Pressure drops within the system
should be avoided as this is counterproductive to certain cell
viability. Reproducibility and homogeneity should be enhanced while
maintaining manual operation and cost at an absolute minimum. A
tortuous path for cells and cell culture media to travel is needed
in conjunction with a spacer layer that facilitates that path along
and parallel to the spacer layer and cell immobilization
layers.
SUMMARY OF THE INVENTION
[0008] In a first aspect, the present invention provides a cell
growth matrix assembly or structured cell growth matrix.
[0009] In a second aspect, the present invention provides for the
use of the cell growth matrix assembly or matrix according to any
embodiment of the invention for growing cells.
[0010] In a third aspect, the present invention provides a
bioreactor comprising a cell growth matrix assembly or matrix
according to any embodiment of the invention.
[0011] The cell growth matrix assembly or matrix of the current
invention present several advantages compared to those known in the
prior art. By making use of a structured assembly, variability
during the cell growth process is omitted or minimalized. The cell
culture surface can be easily and accurately determined thanks to
the known cell culture surface and the number of the immobilization
layers. The cell culture surface to be placed inside a bioreactor
can also be easily adapted without modification of the matrix
and/or the bioreactor design.
[0012] The matrix provides improved and organized cell and cell
culture medium flow inside a bioreactor.
[0013] Furthermore, the structured cell growth matrix provides
efficient packing inside a bioreactor thereby optimizing the use of
the inner space and increasing the cell growth surface inside said
bioreactor. The matrix thereby provides high cell density culture
surface. Moreover, thanks to the structured design of the matrix,
consistent and reproducible production of bioreactors containing
said matrix is facilitated.
DESCRIPTION OF FIGURES
[0014] FIGS. 1A and 1B show cross sectional views of portions of a
matrix assembly according to two embodiments of the current
invention.
[0015] FIG. 2A to D shows non-limitative examples of a matrix
assembly according to embodiments of the current invention.
[0016] FIG. 3A to B show a perspective and a top view of a matrix
assembly partially rolled according to an embodiment of the
invention; and an embodiment of an assembly in tightly packed
configuration.
[0017] FIG. 3C shows a top view of a matrix fully rolled, according
to an embodiment of the current invention.
[0018] FIGS. 4A to 4D show examples of mesh spacer layer structures
according to an embodiment of the current invention.
[0019] FIG. 5 shows a bioreactor chamber provided with a matrix
assembly according to an embodiment of the current invention, in
which fluid and cells flow axially along the surfaces of the spacer
layers and immobilization layers between the top and bottom or
bottom and top.
[0020] FIG. 6 shows a side view of a rotating bioreactor provided
with a matrix assembly according to an embodiment of the current
invention, rotating around its axis when in use.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention concerns a cell growth matrix
assembly, the use of the latter for cell growth and a bioreactor
comprising said matrix assembly.
[0022] Unless otherwise defined, all terms used in disclosing the
invention, including technical and scientific terms, have the
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs. By means of further guidance, term
definitions are included to better appreciate the teaching of the
present invention.
[0023] As used herein, the following terms have the following
meanings:
[0024] "A", "an", and "the" as used herein refers to both singular
and plural referents unless the context clearly dictates otherwise.
By way of example, "a compartment" refers to one or more than one
compartment.
[0025] "About" as used herein referring to a measurable value such
as a parameter, an amount, a temporal duration, and the like, is
meant to encompass variations of +/-20% or less, preferably +/-10%
or less, more preferably +/-5% or less, even more preferably +/-1%
or less, and still more preferably +/-0.1% or less of and from the
specified value, in so far such variations are appropriate to
perform in the disclosed invention. However, it is to be understood
that the value to which the modifier "about" refers is itself also
specifically disclosed.
[0026] "Comprise," "comprising," and "comprises" and "comprised of"
as used herein are synonymous with "include", "including",
"includes" or "contain", "containing", "contains" and are inclusive
or open-ended terms that specifies the presence of what follows
e.g. component and do not exclude or preclude the presence of
additional, non-recited components, features, element, members,
steps, known in the art or disclosed therein.
[0027] The recitation of numerical ranges by endpoints includes all
numbers and fractions subsumed within that range, as well as the
recited endpoints.
[0028] The terms assembly, matrix assembly and matrix are used
interchangeable throughout the text.
[0029] In a first aspect, the current invention provides for a cell
growth matrix assembly, which comprises one or more cell
immobilization layers having a surface which allows cells to adhere
and grow upon and forming a cell immobilization section. Adjacent
to these cell immobilization layers are one or more spacer layers,
including a structure which forms a spacer section allowing passage
of cells and medium through an open but tortuous path whereby the
structure or nature of the spacer layers is chosen such that the
spacer layers create a tortuous, open path for cells and culture
media to travel in parallel to the surface of said spacer and
immobilization layers. The tortuous path or channel formed by the
spacer section creates turbulence which facilitates cell and cell
medium incursion into the immobilization layers.
[0030] Said cell immobilization layers will define a cell
immobilization section, whereas said spacer layers define a spacer
section.
[0031] In further or alternative embodiment, the present invention
provides a structured cell growth matrix comprising one or more
spacer sections and one or more cell immobilization sections. Each
spacer section comprises at least one spacer layer and each cell
immobilization section comprises at least one cell immobilization
layer, wherein the thickness ratio of the immobilization section to
the spacer section is at least 0.1 and the thickness of the spacer
section is at least 0.1 mm. In an embodiment, the spacer section
includes a structure that provides a tortuous path or channel for
cells to travel along the layers.
[0032] For the purpose of the current invention, a tortuous path is
to be understood as a path with directional components that vary
from and along the general path.
[0033] The matrix assembly is designed such that it allows fluid
and cells to flow axially along the surfaces of the spacer layer
and cell immobilization layer in a stationary bioreactor, or in the
case of a rotating bioreactor, tangentially along the surfaces of
both layers. The structure of the spacer layers should be thus
chosen that it creates a tortuous, yet open path in between
immobilization layers, thereby creating turbulence. This turbulence
will drive cells and medium into the immobilization layers.
[0034] It will be understood by the skilled person that such
tortuous path spacer could be achieved by multiple ways, all
readily known in the art. In one embodiment, the spacer layer is
comprised of a bearing structure or spherical, near-spherical or
egg-shaped objects such as beads, packed as a three-dimensional
structure on top of each other (see FIG. 1B). A bearing structure
is to be understood as a structure formed of balls or spheres
optionally fixed to a surface support, which form a tortuous path
through which fluid may flow.
[0035] In another embodiment, the spacer layer may be a mesh or
comprises a mesh structure. For the purpose of the current
invention, mesh structure or mesh is to be understood as a
structure comprising a network or web-like pattern of filament,
wire or thread, said network defines pores, openings or
perforations formed of a three dimensional weave. Examples of mesh
structures are given in FIGS. 4A to 4D. It will be understood that
these cannot be seen as limitative to the current invention.
[0036] The cell growth matrix assembly or matrix according to the
current invention provides for a substrate that allows high density
cell growth. By preference, a high cell density bioreactor allows
for the maximum of cell growth surface in a minimum of volume. The
design of the current cell growth matrix assembly is optimized to
meet these demands. By providing a spacer section adjacent to the
immobilization section, cell and medium flow is ensured. These
spacer sections promote turbulence and allow essentially tangential
flow in between cell adherence sections, in addition to random
perpendicular flow through the immobilization layers. This
increases cells adherence to the immobilization layers of the
immobilization sections.
[0037] Additionally, the cells are more homogenously distributed
inside the bioreactor thanks to the spacer sections which provide
space for cell movement until adherence, and which prevents the
immobilization sections from acting as filters. The spacer further
allows improved removal of toxic metabolites by providing space for
the movement of said metabolites.
[0038] The spacer section further allows better distribution of the
culture medium and thereby of the nutrients inside the bioreactor.
All cells inside the bioreactor are equally provided with culture
medium. Given the structured design of the matrix, preferential
culture medium route will not be created inside a bioreactor. This
is an improvement over the non-structured matrixes of the prior
art, which generate preferential culture medium routes when used in
a bioreactor as the non-structured carriers move under the
influence of the culture medium flow. The result is a
non-homogeneous cell distribution.
[0039] In order to ensure a minimal of pressure drop and volume
loss, the spacer section is comprised of a structure including a
tortuous path for cell and fluid flow. In one embodiment, the
structure is a mesh. By using a mesh structure for spacer section,
the thickness of the physical barrier is kept to an absolute
minimum thereby ensuring constant pressure and yet still allowing
sufficient space between the neighboring immobilization
sections.
[0040] In an embodiment the structure or mesh size of the spacer
layer will be between 0.05 mm to 5 mm. The choice of the structure
or mesh size is important as it again influences the balance
between providing a sufficient barrier between the immobilization
sections thereby allowing cell and medium passage, whilst ensuring
adequate pressure in the system and achieving a high cell density.
The openings in the mesh or grid can be of any shape, such as
round, elliptical, square, or rectangular. In an embodiment, the
size of the openings is at least 0.05 mm, at least 0.06 mm, at
least 0.08 mm, at least 0.1 mm, at least 0.15 mm, at least 0.2 mm
or at least 0.25 mm. In another or further embodiment, the size of
the openings is at most 5 mm, at most 4.5 mm, at most 4 mm, at most
3.5 mm, at most 3 mm or any value comprised in between the
aforementioned values.
[0041] It will be understood that the ratio of the spacer section,
which is built from one or more spacer layers will have an impact
on the functioning of the matrix assembly. In an embodiment the
thickness of the spacer section is at least 0.1 mm, more preferably
between 0.1 mm and 5 mm, more preferably between 0.2 mm and 1 mm.
In an embodiment, the thickness of the spacer section is between
0.25 and 0.6 mm, such as 0.4 mm or 0.5 mm.
[0042] In an embodiment the thickness in mm of the spacer section
is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least
0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at
least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at
least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9,
at least 2, at least 2.1, at least 2.2, at least 2.3, at least 2.4,
at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least
2.9, at least 3, at least 3.1, at least 3.2, at least 3.4, at least
3.5, at least 3.6, at least 3.7, at least 3.8, at least 3.9, at
least 4, at least 4.1, at least 4.2, at least 4.3, at least 4.4, at
least 4.5, at least 4.6, at least 4.7, at least 4.8, at least 4.9,
at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at
least 7.5, at least 8, at least 8.5, at least 9, at least 9.5, at
least 10 or any value comprised between the aforementioned
values.
[0043] In a further or alternative embodiment, the thickness in mm
of the spacer section is at most 1000, at most 950, at most 900, at
most 850, at most 800, at most 750, at most 700, at most 650, at
most 600, at most 550, at most 500, at most 450, at most 400, at
most 350, at most 300, at most 250, at most 200, at most 190, at
most 180, at most 170, at most 160, at most 150, at most 140, at
most 130, at most 120, at most 110, at most 100, at most 95, at
most 90, at most 85, at most 80, at most 75, at most 70, at most
65, at most 60, at most 55, at most 50, at most 45, at most 40, at
most 35, at most 30 at most 25, at most 20, at most 15, at most 12
or any value comprised between the aforementioned values.
[0044] The thickness of the cell immobilization section will
equally be of importance to the functioning of the system. A cell
immobilization section which is too thick will result in a poorly
populated area, whereas a section which is too thin will have a
negative impact on the available cell growth surface, again
negatively influencing cell growth. By preference, the thickness of
the immobilization section will be between 0.1 mm and 15 mm, more
preferably between 0.1 mm and 10 mm, even more preferably between
0.1 and 5 mm, or between 0.1 mm and 1 mm.
[0045] There is a need for a balance between sufficient cell and
medium flow between the cell growth surfaces and a sufficiently
large cell surface for cell growth. The inventors of the current
invention have found that ideally, the ratio between the thickness
of the cell immobilization section to the spacer section should be
at least 0.1, and more preferably between 0.1 and 5, even more
preferably between 0.1 and 2, such as e.g. 1:1. As such, the needs
of the system are met.
[0046] In an embodiment, the thickness ratio of the immobilization
section to the spacer section is at least 0.1, at least 0.2, at
least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7,
at least 0.8, at least 0.9, at least 1, at least 1.1, at least 1.2,
at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least
1.7, at least 1.8, at least 1.9, at least 2, at least 2.1, at least
2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at
least 2.7, at least 2.8, at least 2.9, at least 3, at least 4, at
least, 5, at least 6, at least 7, at least 8, at least 9, at least
10 or any value comprised between the aforementioned values. In a
further or alternative embodiment, the thickness ratio of the
immobilization section to the spacer section is at most 50, at most
45, at most 40, at most 35, at most 30, at most 25, at most 20, at
most 19, at most 18, at most 17, at most 16, at most 15, at most
14, at most 13, at most 12, at most 11 or any value comprised
between the aforementioned values.
[0047] The spacer layers and/or the cell immobilization layers
which make up respectively the spacer section and the
immobilization section in the matrix assembly are preferably made
of a biocompatible polymer selected from polyester, polyethylene,
polypropylene, polyamide, plasma treated polyethylene, plasma
treated polyester, plasma treated polypropylene or plasma treated
polyamide. Said layers can be hydrophilic or hydrophobic. The cell
immobilization layers are preferably hydrophilic.
[0048] The thickness of both layers will advantageously be between
0.05 mm and 3 mm, more preferably between 0.1 and 2 mm or between
0.1 and 1 mm.
[0049] Suitable material for the cell immobilization layer may be a
woven or nonwoven material. By preference, a nonwoven material is
used. A nonwoven, contrary to a woven material, is a fabric which
is not created by weaving or knitting and does not require
converting the fibers to yarn. Nonwovens are broadly defined as
sheet or web structures bonded together by entangling fiber or
filaments (and by perforating films) mechanically, thermally or
chemically. The nature of the nonwoven material used in the current
application may be of any origin, either comprising of natural
fibers or synthetic fibers. By preference, the nonwoven is made of
a polymer, such as polyester or polypropylene. The cell
immobilization layers used in the current invention may be chosen
from a polyethyleentereftalate nonwoven. The nonwoven material may
be plasma treated to enhance cell adherence and flow.
[0050] The spacer layers may consist of a (biocompatible) polymer
with mesh size as described above. In one embodiment, the spacer
layer is a synthetic woven fabric or structure. In another
embodiment, the spacer layer is a bearing structure. Such structure
may be produced from a biopolymer (e.g. alginate). Other suitable
material for this purpose is silica, polystyrene, agarose, styrene
divinylbenzene, polyacrylonitrile or latex.
[0051] The spacer layer may be gamma irradiated in order to reduce
bioburden.
[0052] The design of the matrix assembly can take many forms
depending on the application and type of bioreactor.
[0053] In an embodiment of the current invention, the
immobilization section and spacer section are alternately
positioned. Alternately positioned means that each spacer section
is followed by a cell immobilization section which is itself
followed by a spacer section. The alternately positioned sections
may alternate in vertical position as shown in the figures (see
further) or in a horizontal position according to the use of the
matrix and/or to the bioreactor in which the matrix will be
introduced.
[0054] In this embodiment, one or more layers of cell
immobilization layers are superimposed on one or more spacer layers
(or vice versa). This configuration may be repeated several times
if deemed required in order creating a stack of several
immobilization and spacer sections. Ideally, the end configuration
may comprise between 1 and 500 alternations is of the above
described layering. The stacked layers may be positioned in a frame
or cassette or sealed/connected at their circumference. In another
embodiment, the achieved stack can be rolled around an axis or core
to achieve a spiral configuration.
[0055] The amount of layers used in both the immobilization section
and spacer section can be chosen based on the application,
characteristics of the layers (dimensions, size, etc.)
[0056] and desired result. Hence, the amount of layers within
either immobilization section or spacer section may be between 1
and 20, more preferably between 1 and 10, even more preferably
between 1 and 5.
[0057] As mentioned, the presence of the spacer sections creates
space inside the matrix through which the culture medium flows.
This provides improved circulation of the culture medium through
the matrix thereby reaching all cultured cells. This effect is even
more enhanced in the embodiment wherein the spacer section
comprises one spacer layer and the immobilization section comprises
two immobilization layers. The culture medium flowing inside the
matrix via the spacer sections is tangentially oriented with
respect to the cell immobilization sections.
[0058] The spacer sections improve the rigidity of the matrix
thanks to the rigidity of the spacer layers. The matrix according
to any embodiment of the invention can be compressed by any method
known to the person skilled in the art. The size of the compressed
matrix is reduced by maximum 20%, preferably maximum 15%, more
preferably maximum 10% compared to the size of the non-compressed
matrix.
[0059] In an embodiment, one surface of at least one spacer layer
or section is at least partially free from coverage by any cell
immobilization layers or other layer or section. The non-covered
layer section or layer is designed to be positioned in contact with
the inner wall of any bioreactor. This design allows preventing
plunger effect inside the bioreactor.
[0060] In an alternative embodiment of the current invention, the
matrix assembly is comprised of a configuration of one or more cell
immobilization layers as described above forming an immobilization
section and one or more spacer layers as described above forming a
spacer section, positioned adjacent to (e.g. above and/or under)
said cell immobilization layers. Optionally, the layering may be
repeated to form an alternated, stacked configuration of one or
more cell immobilization layers and one or more spacer layers. Said
resulting configuration is subsequently spiral- or concentrically
wound along an axis or core. The thickness of the spacer section
may be between 0.1 mm and 5 mm, more preferably between 0.2 mm and
1 mm, whereas the thickness of the immobilization section may be
between 0.1 and 5 mm. The ratio of thickness of the immobilization
section over the thickness of the spacer section is preferably
between 1:2 to 2:1, most preferably 1:1. While the amount of layers
to achieve this may vary based on the characteristics of the layers
used and thus freely chosen as described above, it was found that a
good result was achieved when two immobilization layers are used
and one spacer layer. The thickness of the spacer and
immobilization layers is between 0.05 mm and 3 mm, more preferably
between 0.1 and 2 mm or between 0.1 and 1 mm. While the material of
the immobilization and spacer layer should not be construed as
limitative, the immobilization layer is preferably made from a
woven or nonwoven fabric or material as described above for the
other embodiments. Said spacer layer may also be formed of a woven
or nonwoven material and may include a mesh structure.
[0061] By preference, the outer layer of the assembly, being the
side facing the wall of the bioreactor when placed within the
bioreactor will be a spacer layer. This prevents cell growth
against the wall of the reactor. Such bioreactors may be heated and
cell growth in this area could compromise cell yield and/or
quality. In a further embodiment, several spacer layers are
provided at the outer border of the assembly, again to ensure good
insulation. In another or further embodiment, an insulation layer
which is made of another material than the spacer layer is
provided.
[0062] The assembly of the matrix is simple and repeatable compared
to those of the prior art. The invention further offers scalable
matrixes by offering the possibility of easily producing matrixes
having specific dimensions and/or cells growth surface. The
scalability of the assembly/matrix of the invention has no impact
on the homogeneity and/or the quality of the culture medium flow
provided.
[0063] In another aspect, the present invention provides for the
use of the assembly/matrix according to any embodiment described
above for growing cells.
[0064] In another aspect, the present invention provides a
bioreactor comprising an assembly or matrix according to any
embodiment described above. The bioreactor might comprise more than
one assembly or matrix. In a further embodiment, the
assembly/matrix and/or the bioreactor comprising said matrix are
single use. The assembly/matrix can be positioned vertically or
horizontally in the bioreactor.
[0065] Preferably, the assembly/matrix represents at least 10%, at
least 20%, at least 30%, at least 40% or at least 50% of the
bioreactor inner space. Said matrix represents at most 100%, at
most 90%, at most 80%, at most 70% or at most 60% of the bioreactor
inner space.
[0066] The bioreactor can be any type of bioreactor known to the
person skilled in the art such as perfusion bioreactor, wave
bioreactor, cylindrical bioreactor, rotating bioreactor, bag
bioreactor, moving bed bioreactor, packed bed bioreactor, fibrous
bioreactor, membrane bioreactor, batch bioreactor, or continuous
bioreactor. The bioreactor can be of any shape and can be made from
any material, for example, stainless steel, glass, or plastic.
[0067] It is supposed that the present invention is not restricted
to any form of realization described previously and that some
modifications can be added to the presented example without
reappraisal of the appended claims.
FIGURES
[0068] FIGS. 1A and 1B show cross-sectional views of portions of a
matrix assembly according to two embodiments of the current
invention. By providing one or more spacer layers 3 or 3' between
one or more cell immobilization layers 2 (forming the
immobilization section 10), turbulence (depicted with black arrows)
and a random perpendicular flow (horizontal open arrows) is
promoted. The assembly organises a fluid path which allows
sufficient flow distribution when used within a fixed bed. By this
design, liquid flow homogeneity is ensured and as a consequence,
cells are equally homogenously distributed over the assembly. By
providing a spacer creating a tortured, open path, cells and medium
flow is allowed along the surface of both layers. The turbulence
will push cells and medium deeper into the cell immobilization
layers. Overall, the design promotes reproducibility and
homogeneity when used for cell production.
[0069] FIG. 1A depicts an embodiment wherein the spacer section 11
is comprised of one or more spacer layers 3 which are made of a
mesh fabric. Examples of possible mesh fabrics which can be used
for this purpose are shown in FIGS. 4A to 4D, showing various forms
of openings.
[0070] FIG. 1B depicts an alternative embodiment wherein the spacer
section is made of spherical or near-spherical objects such as
beads.
[0071] Both the mesh structure and the beads are examples of
structures or layers which providing a tortured, open path as
described above.
[0072] FIGS. 2A to 2D show possible arrangements of the number of
immobilization layers versus spacer layers which respectively make
up the cell immobilizations section 10 and spacer section 11.
[0073] The thickness of each section corresponds to the sum of the
thickness of the layers contained therein. In a one embodiment, the
spacer section 11 comprises one spacer layer and the cell
immobilization section 10 comprises two cell immobilization layers
as shown in FIGS. 2A and 2B. In this configuration, at least one
surface of each immobilization layer is in contact with one surface
of the spacer layer. FIGS. 2C and 2D show alternative examples,
wherein the spacer section 11 comprises one spacer layer and the
immobilization section comprises three immobilization layers.
[0074] The spacer sections and the cell immobilization section
might be of any shape and might have similar of different
dimensions. The matrix can be formed by alternating the different
sections thereby obtaining a three dimension (3D) matrix. Said
matrix can have any geometrical shape such cylindrical, triangular,
rectangular or any irregular 3D shape. The matrix can also have
other shapes obtained by further shaping the 3D matrix such as
rolling the 3D matrix to obtain a spiral.
[0075] An example of a rolled matrix is shown in FIGS. 3A to 3B
showing respectively a perspective view and a top view of the
rolled matrix. These figures show a matrix or assembly which is
"loosely" rolled to exemplify the spiral structure. It is to be
understood that the degree of rolling the matrix is variable
according to the use of the assembly and/or the wish of the user.
The assembly or matrix will be tightly rolled to a structure such
as the spiral structure shown in FIG. 3C.
[0076] FIG. 3C shows an embodiment of the current invention,
whereby one or more cell immobilization layers 2 are adjacent to
one or more spacer layers 3 made from a mesh structure and whereby
the layering may optionally be repeated several times to achieve a
stacked configuration. Examples of mesh structures are given in
FIGS. 4A to 4D (enlarged view). Preferably, the mesh structure
included in spacer layers of the current invention forms a tortuous
path for cells and fluid to flow when layered between two
immobilization layers. Alternatively, other spacer structures can
be used which form such tortuous paths. The embodiment shown in
FIG. 3C is shown as subsequently spirally or concentrically rolled
along an axis or core whereby all layers are firmly wound. The
diameter of the core, the length and/or amount of the layers will
ultimately define the size of the assembly or matrix. By
preference, the thickness of the layers will be between 0.1 and 5
mm. In the embodiment shown in FIG. 3D, the thickness of the layers
is between 0.25 and 0.7 mm, whereby two layers of cell
immobilization layers are alternated by one spacer layer. By
preference, the outer layer of the resulting spiral configuration
will be one or more spacer layers. Alternatively, an insulating
layer could be provided as outer layer, which differs from both the
spacer and cell immobilization layer. The spacer layers are by
preference a woven mesh fabric with mesh size between 0.05 mm and 5
mm. The cell immobilization layers are preferably a nonwoven
fabric.
[0077] FIGS. 5 and 6 show respectively a stationary bioreactor and
a rotating bioreactor provided with a matrix assembly according to
embodiments of the current invention. In FIGS. 5 and 6, the matrix
as shown in FIG. 3C is included in the bioreactor chambers. In both
embodiments, the flow of cells and medium will be along the path
created by the spacer layers.
[0078] In the embodiment as shown in FIG. 5, culture media and
cells flow through the matrix assembly in the axial direction along
the surface of the spacer and immobilization layers (from top to
bottom or bottom to top).
[0079] FIG. 6 shows a rotating bioreactor whereby the matrix
assembly is partially submerged in medium, and whereby the
bioreactor and matrix are rotated along their axis. In this
embodiment, the flow of medium and cells will be in the tangential
direction along the surfaces of the spacer and immobilization
layers (in the spiral path).
* * * * *