U.S. patent application number 17/423246 was filed with the patent office on 2022-03-03 for functionalized substrate to manipulate cell function and differentiation.
The applicant listed for this patent is KONINKLIJKE PHILIPS N.V.. Invention is credited to LUCAS JOHANNES ANNA MARIA BECKERS, Anja VAN DE STOLPE.
Application Number | 20220064581 17/423246 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-03 |
United States Patent
Application |
20220064581 |
Kind Code |
A1 |
VAN DE STOLPE; Anja ; et
al. |
March 3, 2022 |
FUNCTIONALIZED SUBSTRATE TO MANIPULATE CELL FUNCTION AND
DIFFERENTIATION
Abstract
The invention relates to a scaffold for steering cells into a
predetermined direction of cell functionality, preferably a cell
differentiation scaffold for steering cells into a predetermined
direction of cell differentiation. The scaffold comprises a
polydimethylsiloxane (PDMS)-, or rubber-, or silicone-based
polymeric surface, and one or more cell functionality-inducing
stimuli, preferably one or more cell differentiation-inducing
stimuli, coupled to the polymeric surface.
Inventors: |
VAN DE STOLPE; Anja; (Vught,
NL) ; BECKERS; LUCAS JOHANNES ANNA MARIA; (Veldhoven,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KONINKLIJKE PHILIPS N.V. |
EINDHOVEN |
|
NL |
|
|
Appl. No.: |
17/423246 |
Filed: |
January 17, 2020 |
PCT Filed: |
January 17, 2020 |
PCT NO: |
PCT/EP2020/051125 |
371 Date: |
July 15, 2021 |
International
Class: |
C12M 1/12 20060101
C12M001/12; C12M 3/00 20060101 C12M003/00; C12Q 3/00 20060101
C12Q003/00; C12M 1/00 20060101 C12M001/00; C12M 1/42 20060101
C12M001/42; G16B 5/20 20060101 G16B005/20; C12N 5/071 20060101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 17, 2019 |
EP |
19152226.7 |
Claims
1. A substrate, for steering of cells into a predetermined
direction of cell functionality, the steering of cells consisting
of steering cells into a predetermined direction of cell
differentiation, the substrate comprising: an elastomer material
having coupled thereto one or more cell functionality-inducing
stimuli wherein the cell functionality-inducing stimuli comprise
cell differentiation-inducing stimuli.
2. The substrate according to claim 1, wherein the one or more cell
functionality-inducing stimuli are selected from the group
consisting of: cell receptor ligands, ectracellular matrix modules,
artificial receptor agonists or antagonists, and specific antigens
for T or B cell receptors.
3. The substrate according to claim 1, wherein the one or more cell
functionality-inducing stimuli are cell receptor ligands.
4. The substrate according to claim 1, wherein the cell receptor
ligands are configured for activating signaling through a signaling
pathway selected from the group consisting of: nuclear receptors,
such as ER, AR; progesterone receptors; growth factor pathways,
such as: PI3K-FOXO, JAK-STAT3, MAPK-AP1 pathways; immune pathways,
such as STAT1/2 type I interferon (STAT1/2-1), STAT1/2 type II
interferon (STAT1/2-2) pathways; developmental pathways, such as
Hedgehog, Notch, TGF-.beta., and Wnt signaling pathways; and
inflammatory pathways such as NFkB.
5. The substrate according to claim 1 wherein the elastomer
material comprises one or more polysiloxane polymers or
polybutadiene polymers.
6. The substrate according to claim 1, wherein the one or more cell
functionality induced stimuli coupled to the elastomer material are
obtainable by reacting a functional group of the one or more cell
functionality-induced stimuli with a further reactive group of a
precursor elastomer material using a crosslinking reaction chosen
from the group of bioconjugation reactions.
7. The substrate according to claim 1, wherein the one or more cell
functionality-inducing stimuli are coupled to the elastomer
material via one or more of an amide, amine and ester group.
8. A culture unit comprising a cell culture chamber having therein
a substrate according to claim 1.
9. Use of the substrate or culture unit according to claim 8, for
producing a cell culture tissue or organ.
10. An In vitro method of producing a cell culture, tissue or
organ, the method comprising: (a) determining first culture
conditions that control activity of at least one first signaling
pathway of cells to be cultivated, the activity of the at least one
first signaling pathway being a selected activity to steer the
cells into a first predetermined direction of cell functionality;
and (b) cultivating cells in a culture unit under the first culture
conditions to obtain a cell culture.
11. The In vitro method according to claim 10, further comprising:
(c) inferring activity of the at least one first signaling pathway
in a cell sample obtained from the cell culture; (d) comparing the
inferred activity with the selected activity, and (e) determining
second culture conditions based on the comparison of the inferred
activity with the selected activity.
12. The In vitro method according to claim 11, further comprising
determining second culture conditions based on the comparison of
the inferred activity with the selected activity and wherein the
second culture conditions are determined to (i) reduce deviation of
the inferred activity from the selected activity, or (ii) control a
selected activity of at least a second signaling pathway different
from the at least one first signaling pathway, the selected
activity of the at least one second signaling pathway being
selected to steer the cells into a second predetermined direction
of cell functionality different from the first predetermined
direction of cell functionality.
13. The In vitro method according to claim 10, further comprising:
(f) cultivating the cells in the culture unit under the second
culture conditions; (g) inferring activity of the at least one
first signaling pathway in a cell sample obtained from the cell
culture; (h) comparing the inferred activity with the selected
activity, and (i) determining third culture conditions based on the
comparison of the activity inferred in steps (g) and (h) with the
selected activity.
14. The In vitro method according to claim 9, wherein the cell
functionality is selected from the group consisting of cell
differentiation, maintenance of pluripotency and cellular function,
such as cell division, migration and metabolism, , adhesion,
apoptosis, generating oxidative stress, DNA repair, production of
secretory proteins, production of extracellular matrix proteins,
preferably cell differentiation.
15. The In vitro method according to claim 9, wherein the first
and/or second culture conditions are defined by controlled exposure
of the cells to one or more cell functionality-inducing stimuli
selected from the group consisting of cell receptor ligands,
extracellular matrix molecules, artificial receptor agonists or
antagonists, and specific antigens for T or B cell receptors, the
stimuli controlling activity of the at least one first and/or
second signaling pathway, and/or wherein the one or more cell
functionality-inducing stimuli are provided in the form of one or
more culture units.
16. The In vitro method according to claim 9, wherein the cells are
mouse stem cells, human stem cells, pluripotent stem cells,
mesenchymal stem cells, embryonic stem cells, adult stem cells,
tumor cells, cell lines.
17. A non-transitory storage medium storing instruction that are
executable by a digital processing device to perform the method of
claim 9.
18. A computer program comprising program code means for causing a
digital processing device to perform the method of claim 9, when
the computer program is run on a digital processing device.
19. A system for performing the method of claim 14, the system
comprising (a) one or more culture units, (b) a sensor unit for
inferring an activity of at least one first signaling pathway; (c)
a controller that is configured to compare the inferred activity
with a predetermined activity of the at least one first signaling
pathway, the predetermined activity being selected so to
controllably steer the cells into a first predetermined direction
of cell functionality, and (d) a computer program, wherein,
preferably, the controller is further configured to determine
culture conditions based on the comparison of the inferred activity
with the predetermined activity, the culture conditions being
optionally determined so as to (ii) reduce deviation of the
inferred activity from the predetermined activity or (ii) control
activity of at least one second signaling pathway, the activity of
the at least one second signaling pathway.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to the field of
biotechnology, bioinformatics and related arts. More particularly,
the present invention relates to a substrate or scaffold for
steering cells into a predetermined direction of cell functionality
and also to a culturing unit comprising such substrate or scaffold.
The invention also pertains to an in vitro method of producing a
cell culture, tissue or organ by steering cells into a
predetermined direction of cell functionality. The present
invention further relates to a non-transitory storage medium
storing instruction that are executable by a digital processing
device to perform the aforementioned method. The present invention
further relates to a computer program comprising program code means
for causing a digital processing device to perform the
aforementioned method, when the computer program is run on a
digital processing device. The present invention further relates to
a system for performing the aforementioned method.
BACKGROUND OF THE INVENTION
[0002] Cell function and differentiation during embryonic
development, in adult life, and in disease, is controlled and
steered by around 15 signal transduction pathways, like for example
the Hedgehog, PI3K and estrogen receptor pathways. Pathway activity
is regulated by cell-extrinsic factors among others, including
environmental stimuli, such as small molecules, secreted proteins,
temperature, and oxygen. These stimuli can originate from other
cells within the organism, or they can come from the organism's
environment. Within the organism, cells communicate with each other
by sending and receiving secreted proteins, also known as growth
factors, morphogens, cytokines, or signaling molecules. Receipt of
these signaling molecules triggers intercellular signaling cascades
that ultimately cause changes in transcription or expression of
genes. This process is thought to regulate a vast number of cell
behaviors.
[0003] In higher organisms, intercellular signaling pathways have
the important task of coordinating and regulating cell division.
The pathways ensure that cells divide synchronously and, if
necessary, arrest cell division and enter a resting state. Cellular
communication assumes great importance in the differentiation and
development of an organism. The development of an organism is based
on genetic programs that always utilize inter- and intracellular
signaling pathways. Signal molecules produced by one cell influence
and change the function and morphology of other cells in the
organism. Intercellular signaling pathways are also critical for
the processing of sensory information. External stimuli, such as
optical and acoustic signals, stress, gradients of nutrients, and
so on, are registered in sensory cells and are transmitted to other
cells of the organism via intercellular signaling pathways.
Naturally, a variety of timely and spatially controlled stimuli are
provided to regulate cellular behavior.
[0004] For example, during embryonic development, stem cells
differentiate into all kinds of body cells. The stem cells go
through multiple defined differentiation steps to finally mature
into the fully differentiated cell, like a skin cell, neuron, etc.
During adult life stem cells and progenitor cells reside in organ
niches under highly specific conditions and differentiate to
replace organ cell types that are lost due to physiological
processes (e.g. in the intestinal mucosa) or due to
disease-associated organ damage. In benign and malignant tumors
cells have lost differentiation features, allowing them to start
dividing and metastasizing; this is again orchestrated by
(spatiotemporal) activity of the signal transduction pathways. All
this is orchestrated by highly controlled (spatiotemporal) activity
of signal transduction pathways.
[0005] In other diseases, there is often abnormal activity of
signal transduction pathways, which plays a causal role in the
disease, in its progression and response to therapy.
[0006] Investigation of function of cells and tissues or of a
specific disease to better understand (patho-)physiology and to
develop drugs and therapies (e.g. regenerative medicine) requires
in vitro cell and tissue culture model systems for healthy and/or
diseased cell/tissue. The success of these and other in vitro
applications that reside on cells to acquire a particular cellular
behavior largely depends on the conditions in the immediate
vicinity of the cells. Physical, chemical, and biological control
of cell microenvironment are thus considered to be of crucial
importance for the ability to direct and control cell behavior
spatially and temporally in clinically associated application such
as the production of 3-dimensional tissue engineering
scaffolds.
[0007] A precise control of multiple stimuli in the cell culture
microenvironment, which regulate intracellular signaling and
ultimately cell phenotype, is particularly important in stem cell
culture and differentiation (but similarly important to other
cultures). Recapitulating the in vivo cells/tissue by in vitro
culture is possible, on the premise that the required stimuli
(stimuli) are provided in the correct spatiotemporal manner. The
stimuli may consist of ligands, mostly proteins, that bind to and
activate specific cell membrane receptors, to activate certain
intracellular signal transduction pathways. Activation of such a
pathway leads to a functional change in the cell. Moreover, the
culture surface should mimic the natural in vivo cellular
environment, which is a hurdle to optimally and most efficiently
differentiating and maturing stem cells in the required
directions.
[0008] For already differentiated cells it is generally more
important to influence the function of the cells in a controlled
manner, like cell division, migration, metabolism and the like.
However, the same requirements as given for the differentiation of
stem cells apply.
[0009] While it is difficult for conventional culture systems to
provide such an accurate control in vitro, microfluidic devices are
thought to provide a solution to this problem. For example, in a
review of Zhang et al, published in Future Sci. OA, 2017, 3(2),
FSO187, it is explained that microfluidic devices are ideally
suited for stem cell culture and maintenance by providing the
stimuli to create an in vivo like microenvironment. Advantageously,
microfluidic devices offer the possibility to facilitate
stimulation of cells by both biochemical stimuli and structural
stimuli including well-defined surface features and patterned
scaffolds, mechanical stress and electromagnetic forces, among
others. Moreover, microfluidic devices can control multiple stimuli
simultaneously over space and time with high precision and may
thereby provide an ideal and well-defined platform for stem
cells.
[0010] A disadvantage of known techniques, whether carried out in
conventional culture systems or microfluidic devices, is that they
rely primarily on control of culture conditions. The term control
as used by various authors including Zhang et al, is to be
understood to describe generally the possibility to tune culture
conditions to provide a microenvironment suitable for the
respective purpose. The selection of suitable culture conditions is
based on existing knowledge and/or extensive experience by the
experimenter. With these methods the results are heavily dependent
on the level of existing knowledge and/or experience.
[0011] It would therefore be desirable to have methods that allow a
control of culture conditions that is more robust, in particular
less independent from existing knowledge and/or experience, as well
as means that can be suitably employed in such methods.
SUMMARY OF THE INVENTION
[0012] In accordance with a first aspect of the invention, the
above problem is at least partly solved by a substrate or scaffold,
for steering of cells into a predetermined direction of cell
functionality, the substrate or scaffold comprising an elastomer
material having coupled thereto one or more cell
functionality-inducing stimuli.
[0013] The substrate, which preferably comprises or is a scaffold,
is for steering cells into a predetermined direction of cell
functionality. Such steering can typically be done during
cultivation or culturing of cells. The substrate includes an
elastomeric material to which the one or more cell
functionality-inducing stimuli are coupled. The stimuli are for
aiding or causing the steering. This implies that the stimuli are
preferably at least present at an elastomer surface of the
elastomer material to be available for such steering during
cultivation of cells. The presence of the stimuli at the
elastomeric material surface provides a controlled environment in
that the steering can take place at or near the surface of the
elastomeric material.
[0014] The substrate may be a cell differentiation substrate or the
scaffold may be a cell differentiation scaffold for steering cells
into a predetermined direction of cell differentiation.
[0015] The substrate or scaffold may be part of a culture unit.
There is provided Culture unit comprising a cell culture chamber
having therein a substrate or scaffold according to any one of the
previous claims. The substrate or scaffold is thus arranged to be
partly disposed in the chamber.
[0016] The culture unit may be part of a microfluidic device. Such
device can have other functionalities useful for the cultivation of
cells. For example there may be implemented one or more canals for
transporting fluids and/or gases to and from the substrate for
aiding cultivation of cells.
[0017] The substrate, scaffold, and culture unit are thus designed
to provide a controlled environment for steering cells into a
desired direction of cell functionality during cultivation of
cells.
[0018] In accordance with that purpose there is provided the use of
the substrate or scaffold according to any one of claims 1 to 8, or
the culture unit according to claim 8 for producing a cell culture
tissue or organ.
[0019] Accordingly, a second aspect of the present invention
relates to a method of producing a cell culture, tissue or organ
using steering of cells into a predetermined direction of cell
functionality. The method is defined comprises the steps of: [0020]
(a) determining first culture conditions that control activity of
at least one first signaling pathway of cells to be cultivated, the
activity of the at least one first signaling pathway being selected
so to control cell behavior in a first predetermined manner; [0021]
(b) cultivating cells in a culture unit under the first culture
conditions; and optionally also the steps. Optionally, the method
further comprises [0022] (c) inferring activity of the at least one
first signaling pathway in a cell sample obtained from the cell
culture; and [0023] (d) comparing the inferred activity with the
selected activity.
[0024] The inventors of the present invention found that despite of
steadily increasing knowledge and experience with regard to stimuli
required for a cell to acquire a particular cell behavior, it is
difficult to consistently steer cells into the same direction. The
level of knowledge and/or experience has a direct effect on the
obtained result. If corrections to the culture conditions during
cultivation are made, then these are based on deviations of actual
culture condition from the culture conditions which according to
existing knowledge and/or experience lead to the desired cell
behavior. It is not taken into account that the existing knowledge
and/or experience may be insufficient or incorrect and hence that
the culture conditions to be assumed to be suitable are sub-optimal
or even wrong.
[0025] With the present invention a robust method is provided that
can be controlled so that cells may reproducibly adopt a particular
cellular behavior that is less dependent from existing knowledge
and/or experience as known methods. The method of the present
invention allows to directly determine whether the cells indeed
react to the culture conditions determined in step (a) in the
expected manner. Therefore, in the first step (a) culture
conditions are determined that are known or at least assumed to
steer a particular cell (i.e. cell type) towards a desired cell
behavior. In step (b), cells of the same cell type as in (a) are
cultivated to provide a cell culture. Pathway activity is inferred
in step (c). Comparing the inferred activity with the activity
selected so to control cell behavior in a first predetermined
manner (i.e. the selected activity) of the at least one first
signaling pathway during cultivation allows to monitor cell
response on the level of the cell culture, monitor progress of the
cell culture to acquire a particular cellular behavior, confirm or
decline that the cell culture has acquired the desired cell
behavior or steer into the desired direction, and the like. More
particularly, the present invention allows to draw conclusions with
regard to cell behavior by preferably quantitatively inferring
activity of signaling pathway(s) that mediate the respective cell
behavior. For example, differentiation of stem cells to certain
tissue may involve activation of one or more pathways. Inferring
activity of said one or more pathways allows to safeguard that the
respective signaling pathway(s) is/are indeed active. Information,
in particular quantitative information, on the pathway activity can
be advantageously used for the purposes of the present invention as
a basis for minimizing deviations from the desired cell behavior so
that cell behavior can be precisely controlled, and cells can be
steered into the desired direction. The present invention thus
provides a method which enables cell cultivation in a more
controllable fashion than it is possible with existing methods.
[0026] According to a fourth aspect, the problem is solved by a
non-transitory storage medium storing instruction that are
executable by a digital processing device to perform the method of
the invention.
[0027] According to a fifth aspect, the problem is solved a
computer program comprising program code means for causing a
digital processing device to perform the method of the invention,
when the computer program is run on a digital processing
device.
[0028] According to a sixth, aspect the problem is solved a system
for performing the method of the invention, the system comprising
[0029] (a) a culture unit or a scaffold, [0030] (b) a sensor unit
for inferring an activity of at least one first signaling pathway;
[0031] (c) a controller that is configured to compare the inferred
activity with a selected activity of the at least one first
signaling pathway, the selected activity being selected so to
control cell behavior in a first predetermined manner of the
invention or a computer program of the invention.
[0032] Further advantages will be apparent to those of ordinary
skill in the art upon reading and understanding the detailed
description provided herein below.
[0033] This application describes several preferred embodiments.
Modifications and alterations may occur to others upon reading and
understanding the preceding and following description. It is
intended that the application is construed as including all such
modifications and alterations insofar as they come within the scope
of the appended claims or equivalents thereof.
[0034] Other variations to the disclosed embodiments can be
understood and effected by those skilled in the art in practicing
the claimed invention, from a study of the disclosure, and the
appended claims.
[0035] It shall be understood that the substrate or scaffold of
claim 1, the culture unit of claim 8, the use of claim 9, the
method of claim 10, the non-transitory storage medium of claim 17,
the computer program of claim 18 and the system of claim 19 have
similar and/or identical preferred embodiments or make use of
corresponding features, in particular, as defined in the dependent
claims.
[0036] It shall be understood that a preferred embodiment of the
present invention can also be any combination of the dependent
claims or above embodiments with the respective independent
claim.
[0037] These and other aspects of the invention will be apparent
from and elucidated with reference to the embodiments described
hereinafter.
BRIEF DESCRIPTION OF THE DRAWING
[0038] In the following drawings:
[0039] FIG. 1 exemplarily illustrates pathway activity during
differentiation of human stem cells (Human embryonic stem cells,
undifferentiated; hESC undiff) to endoderm by SR-1 induction (SR-1
ind.). Mesoderm (MD) is formed by AFBLy-differentiation (AFBLy-diff
hESC) of hESCs (log2odd values shown after 3 days (3 d)). The
pathway activities were obtained by Philips pathway analysis on
Affymetrix U133 Plus2.0 microarray data available in the publicly
available GEO dataset GSE52658.
[0040] A. Philips pathway activities measured per differentiation
step are indicated next to the annotation (e.g. anterior primitive
streak (APS), definitive endoderm (DE), mesoderm (MD), anterior
foregut (lungs, thyroid) (AF), posterior foregut (pancreas, liver)
(PF), midgut/hindgut (intestines) (M/H), etc.) per individual
sample, as log2odds score of the respective pathway activity,
respectively PI3K-FOXO, Hedgehog (HH), TGF-.beta., Wnt, Notch,
STAT3 pathways; names of the pathways are listed in the box on top
of the activity scores.
[0041] The pathway activities (also referred to as pathway activity
scores) were determined on a log2odds scale; accordingly, the
indicated values are log2odds values.
[0042] B. Overview picture showing sequential differentiation steps
during which specific pathways are activated (by SR-1 induction
(SR-1 ind.) after 1 day (1 d), 3 days (3 d), and 7 days (7 d)),
necessary to obtain the respective step in differentiation;
signaling pathway activities are indicated as log2 odds values per
individual sample for respectively PI3K-FOXO, Hedgehog, TGF-.beta.,
Wnt, Notch, STAT3 pathways. Human embryonic stem cells (hESC) can
be differentiated into various cell-types under the control of
specific signals. Signaling pathway activity is a readout of such
signals and measuring pathway activity can be a useful tool to
assess the differentiation/maturation status of cultured cells.
hESCs can be differentiated into various cell types (in this
example in FIG. 1B: anterior foregut (lungs, thyroid) (AF),
posterior foregut (pancreas, liver) (PF), (M/H) midgut and hindgut
(intestines) (M/H)) by passing through various differentiation
steps (in this example in FIG. 1B: anterior primitive streak (APS)
and definitive endoderm (DE)), as depicted by arrows in FIG. 1B.
For example, anterior foregut (AF) is formed from anterior
primitive streak via definitive endoderm. Mesoderm (MD) is formed
by AFBLy-differentiation (AFBLy-diff. hESC) of hESCs (log2odd
values shown after 3 days (3 d)). Signaling pathway activity was
measured using Philips signaling pathway analysis for each cell
type, resulting in specific pathway signatures.
[0043] FIG. 2 exemplarily shows the results of a differentiation
experiment carried out on non-functionalized surface (A.) and
functionalized surface coated with TGFb1 (B.), on which WPMY-1
cells were seeded. Functionalization was carried out with EDC/NHS
as disclosed herein. In the TGFb1 coating experiment, WPMY1
fibroblast cells were seeded and cultured on DMEM medium with 10%
FBS on functionalized surface (SIL-IM-F 0.5%, silicone substrate
containing linoleic acid as described herein and in international
patent application PCT/EP2018/068318 referred to above), which was
according to described protocol coated with TGFb1 by incubation
with 10 ug TGFb1 (SIL-IM-F 0.5%-TGFb1). The non-functionalized
surface (A.) was similarly incubated with TGFb1 and served as a
control. Cells bind in a specific manner to the TGF-.beta.-coated
surface, which binding is assumed to be dependent on the cells'
expression of TGF-.beta. receptors. On the non-coated surface cells
bind in a specific manner, independent of expression of specific
receptors, like the TGF-.beta. receptor. Fewer cells bind to the
TGF-.beta.-coated surface, and cell morphology changes, probably
caused by the TGF-.beta. induced activation of the TGF-.beta.
signal transduction pathway (see experiment using TGF-(3 as an
example described further below).
[0044] FIG. 3 exemplarily shows an independent cell culture
experiment in which the concentration of TGFb1 has been varied on 3
different surfaces. A tissue culture-treated plastic surface as a
control (CT), a non-functionalized silicone surface (NFS) and a
functionalized silicone surface (FS) were incubated with different
concentrations of TGFb1, 0 ng/ml, 10 ng/ml and 1000 ng/ml
(indicated in FIG. 3 as respectively NO, 10 ng and 1 microgram). On
the y-axis is shown the TGF-.beta. pathway activity score.
[0045] FIG. 4 exemplarily shows another independent cell culture
experiment in which JAG1 was used as a NOTCH receptor ligand
coupled to polystyrene (PS), modified silicone (MS) and regular
silicone (S) culture plates in order to specifically activate the
Notch pathway in cell cultures. The ligand-coupled polystyrene
culture plate served as a positive control, the non-modified
silicone culture plate served as negative control. Each
ligand-involved experiment (right bar of each of the three pairs)
is accompanied by a respective experiment without ligand (left bar
of each of the three pairs). Pathway activity is indicated as
pathway score, whereby the higher the score the higher the Notch
pathway activity.
DETAILED DESCRIPTION OF EMBODIMENTS
[0046] The following embodiments merely illustrate particularly
preferred methods and selected aspects in connection therewith. The
teaching provided herein may be used for constructing several test
methods, test systems and/or test kits, e.g., to produce a cell
culture, tissue and/or an organ. The following embodiments are not
to be construed as limiting the scope of the present invention.
[0047] The extracellular microenvironment plays a significant role
in influencing cellular behavior including maintenance of potency,
cellular attachment, proliferation, metabolization,
lineage-specific differentiation and remodeling. By manipulating
the culture conditions in which cells including stem cells are
cultivated, it is possible to influence cell behavior, restrict
differentiation pathways and thereby produce cell cultures enriched
in lineage-specific precursors, particular tissues or organs in
vitro among others. Based on this understanding, one aspect of the
invention relates to a substrate such as for example a scaffold,
for steering cells into a predetermined direction of cell
functionality.
[0048] The elastomer material comprising (i.e. binding) the one or
more stimuli may be denoted as scaffold or cell culturing scaffold
in correspondence to the terminology used in the aforementioned
international patent application published under number WO
2019/015988. Such a cell culturing scaffold can be manufactured in
a straightforward manner by a simple coupling reaction of the one
or more stimuli to reactive groups such as for example carboxylic
acid groups) of the elastomer material or a precursor elastomer
material, thereby leading to a cell culturing scaffold having a
substantially homogeneous distribution of the one or more stimuli
across a surface of the material.
[0049] In an embodiment the substrate or scaffold is a cell
differentiation scaffold for steering cells into a predetermined
direction of cell differentiation. The scaffold comprises a
polydimethylsiloxane (PDMS)-, or rubber-, or silicone-based
polymeric surface, and one or more cell functionality-inducing
stimuli, preferably one or more cell differentiation-inducing
stimuli, coupled to the polymeric surface.
[0050] The term "cell functionality", also referred to herein as
"cell behavior" or "cellular behavior", denotes a functional or
morphological response of a cell or cell culture to the culture
conditions applied. Typical functional responses that may be
induced include, for example, cell division, cell growth,
differentiation, development, maintenance of pluripotency, etc.
Further cell behaviors that can be controlled by the present
invention are described herein.
[0051] The term "cell functionality-inducing stimuli" refers to
culture conditions, as disclosed herein, that are provided in order
to induce a predetermined cell functionality in the cells.
[0052] The one or more cell functionality-inducing stimuli,
preferably differentiation-inducing stimuli are preferably selected
from the group consisting of cell receptor ligands, extracellular
matrix molecules, artificial receptor agonists or antagonists, and
specific antigens for T or B cell receptors. Preferably, the one or
more cell functionality-inducing stimuli, preferably
differentiation-inducing stimuli, are ligands activating signaling
through a signaling pathway selected from the group consisting:
nuclear receptors, such as ER, AR, progesterone receptor; growth
factor pathways, such as PI3K-FOXO, JAK-STAT3, MAPK-AP1 pathways;
immune pathways such as STAT1/2 type I interferon (STAT1/2-1),
STAT1/2 type II interferon (STAT1/2-2) pathways; developmental
pathways, such as Hedgehog, Notch, TGF-.beta., and Wnt signaling
pathways; and inflammatory pathways such as NFkB. The ligand can be
of natural or non-natural (synthetic) origin. The ligand may be an
artificial ligand and/or does not occur in nature as such. The
ligand may be an artificial and/or synthesized protein ligand such
as an activating antibody or peptide.
[0053] A further preferred embodiment relates to a substrate, as
disclosed herein, wherein the one or more cell
functionality-inducing stimuli, preferably differentiation-inducing
stimuli are coupled to the polymeric surface via carboxyl groups.
The substrate or scaffold may be any shape or form. It may be flat
or not. It may have a relief such as corrugated or spiked or other.
The substrate may comprise or consist of a scaffold where scaffold
is intended to imply to provide a support function which may be two
dimensionally or three-dimensionally shaped.
[0054] The substrate or scaffold may be part of a culturing device.
Such device generally includes a culturing chamber comprising the
substrate or scaffold. One or more channels connected to the
chamber may be present to transport fluids from and to the chamber.
The chamber and substrate may be formed within a device body and in
an embodiment there may be one or more covers that are removable
from the body so as to access the chamber from the outside. In a
preferred embodiment, the substrate is between (e.g. stacked)
between a top cover plate and a bottom cover plate. The substrate
may optionally be adapted to adhere to the top cover plate and/or
the bottom cover plate. Many such devices exist and will not be
described here in detail. Examples of how to design culturing units
and microfluidic devices can be found in for example International
application published under number WO 2019015988. Examples of
precursors for manufacture of substrates and scaffolds can also be
found in this publication as will be elucidated in more detail
herein below.
[0055] The scaffold, and the culture unit, preferably microfluidic
device, is designed to provide a controlled environment for
steering cells into a desired direction of cell functionality. In
accordance with this suitability, a third aspect of the present
invention relates to a method of producing a cell culture, tissue
or organ. The method comprises the step (a) of determining first
culture conditions that control activity of at least one first
signaling pathway of cells to be cultivated. The activity of the at
least one first signaling pathway is thereby selected so to
controllably steer the cells to be cultivated in a first
predetermined direction of cell functionality. The phrase "to
control cell behavior in a predetermined manner" is understood the
describe a step, wherein the cells to be cultivated are
controllably steered into a predetermined direction of cell
functionality. The determination in step (a) does not imply an
active experimental step but may simply mean that information is
gathered, e.g. based on in silico predictions, existing knowledge
(for example published literature and/or data banks) and/or
experience, which signaling pathways are active or inactive in the
cells (cell type) of interest in order such cells acquire a
particular cellular behavior (i.e. cell functionality). For
example, it is known that differentiation of certain stem cells
towards specific cell types or tissue involves a cascade of cell
signaling (cf., e.g. Tsai et al, BMC Cell Biol, 2010, 11:76). Based
on this knowledge, it can be determined that these signaling
pathways should be active or inactive in a sequential manner to
arrive at the desired progeny.
[0056] The terms "pathway", "signal transduction pathway" and
"signaling pathway" are used interchangeably herein. The at least
one signaling pathway may mediate cell behavior of the cells to be
cultivated. The term "inferring activity" in the context of pathway
activity herein implies a step in which a dimension corresponding
to the pathway activity is inferred. The dimension may preferably
describe the pathway activity in a semi-quantitative manner,
wherein the activity may be classified according to, for example,
no activity, low activity, high activity, and the like, or in a
quantitative manner, wherein the activity is expressed in the form
of discrete or continuous values, the values corresponding to
levels of activity or degree of pathway activation. In this way, a
semi-quantitative or quantitative comparison in step (d) is
possible. The comparing in step (d) allows to determine whether the
inferred activity corresponds to the selected activity so as to
control cell behavior in the predetermined manner, i.e. to
controllably steer the cells into the predetermined direction of
cell functionality. It can therefore be determined whether the
cultivated cells behave in the desired manner, i.e. steer into the
desired direction of cell functionality, e.g. differentiate to the
desired progeny, maintain pluripotency or the like, and if so the
culture conditions may be maintained or if not they may be changed
so as to correct any observed deviation from the desired behavior
reflected by the pathway activity determined in step (a).
[0057] According to a preferred embodiment, the method further
comprises step (e) of determining second culture conditions based
on the comparison of the inferred activity with the selected
activity. Preferably, the second culture condition is determined so
as to (i) reduce deviation of the inferred activity from the
selected activity. This means, in case the inferred activity of the
at least one signaling pathway does not correspond to the expected
activity, i.e. the activity determined in step (a), the second
culture conditions determined in step (e) may reflect conditions
that are expected to correct any observed deviation from the
desired behavior reflected by the pathway activity determined in
step (a).
[0058] Alternatively, the second culture conditions may be
determined so as to (ii) control activity of at least a second
signaling pathway, the activity of the at least one second
signaling pathway being selected so to control cell behavior in a
second predetermined manner. For example, the inferred activity
corresponds to the expected activity and the second culture
conditions determined in step (e) may be substantial identical to
the first culture conditions determined in step (a). This is useful
in case the cell culture is intended to continue to divide and/or
maintain pluripotency. According to another example, the inferred
activity corresponds to the expected activity and the second
culture conditions are determined in step (e) to control activity
of at least one second signaling pathway of the cells, the activity
of the at least one second signaling pathway being selected so to
control cell behavior in a second predetermined manner, i.e. to
controllably steer the cells into a second predetermined direction
of cell functionality. This may be particularly useful, when
differentiation of the cells to a particular progeny involves a
cascade of signaling pathways that needs to be activated or
deactivated in a sequential manner.
[0059] According to a further preferred embodiment, the method
further comprises the following steps (f) cultivating the cells in
the culture unit under the second culture conditions; and (g)
repeating steps (c) and (d) as described herein. Preferably, the
steps (c) and (d) are repeated in intervals. The intervals may be
set depending on the cells, cultivation conditions and/or desired
cell behavior and may range from a few hours to several weeks.
Suitable intervals for example range from 6 hours to 3 weeks, 6
hours to 2 weeks, 6 hours to 10 days, 6 hours to 7 days, preferably
8 hours to 5 days, more preferably 8 hours to 3 days, in particular
8 hours to 2 days such as once daily.
[0060] Optionally, the method further comprises step (h)
determining third culture conditions based on the comparison of the
activity inferred in step (g) with the selected activity. In this
manner, the method enables feedback control of the cell behavior
based on pathway activity. The optional step (h) may for example
reflect conditions to influence activity of at least one third
signaling pathway, for example a third signaling pathway involved
in a cascade of multiple signaling pathways required for
differentiation of the cells to a particular progeny.
[0061] With the method of the present invention cell behavior that
is mediated by one or more signaling pathways can be controlled.
The cell behavior that can be influenced is not particularly
limited and includes differentiation, maintenance of pluripotency
and cellular function, such as cell division, migration and
metabolism, chemotaxis, adhesion, apoptosis, generating oxidative
stress, DNA repair, production of secretory proteins, production of
extracellular matrix proteins and the like.
Stimuli
[0062] According to a preferred embodiment, the first, second
and/or third, etc., culture conditions are defined by controlled
exposure of the cells to one or more cell-functionality inducing
stimuli, wherein the stimuli control activity of the at least one
first and/or second signaling pathway. In other terms, the cell
culture is exposed to well-defined stimuli that may activate or
deactivate the respective signaling pathway(s). Suitable stimuli
include both naturally occurring substances but also non-natural
substances such as artificial proteins or peptides, drugs that
activate or modify pathway activity by binding to a surface
receptor, such as antibodies like trastuzumab and antigen peptides
that bind to T cell or B cell receptors and initiate cell division
through the PI3K pathway. Accordingly, it is preferred that the one
or more cell-functionality inducing stimuli are selected from the
group of cell receptor ligands, extracellular matrix molecules,
artificial receptor agonists or antagonists, or specific antigens
for T or B cell receptors.
[0063] According to a preferred embodiment, the one or more
cell-functionality inducing stimuli are provided in the form of a
scaffold or culture unit, as defined herein.
[0064] Accordingly, the one or more cell-functionality inducing
stimuli may be coupled to the polymeric surface of the scaffold or
culture unit. Coupling the one or more stimuli to a surface in the
correct three-dimensional conformation in the immediate
microenvironment of the cells more closely reflect in vivo
situation, where ligands are often expressed on the outside of cell
membrane of an adjacent cell or bound to the extracellular matrix
surrounding the cells, than by providing them in solution flowing
along the cells attached to the surface. This embodiment may render
many ligands very effective.
[0065] The one or more cell-functionality inducing stimuli are
preferably provided in a definite spatial and/or temporal manner.
To provide the one or more stimuli in a particular spatial
distribution the surface may be patterned with different types of
stimuli. For example, two different cell types with different
stimuli requirements may be aligned. In addition, the concentration
of stimuli may be varied and/or adapted to the particular need. For
example, a low concentration of a particular stimuli may induce
cell differentiation, whereas a high concentration of the same
stimuli induces cell division. The TGF-.beta. pathway as a typical
example dictates cell fate in a concentration-dependent manner. In
addition or alternatively to variations of the concentration,
concentration gradients may be envisaged. Mixtures of stimuli may
be provided when different receptors need to be activated to obtain
the desired effect in the cells. An example would be use of a
growth factor ligand in combination with TGF-.beta..
Surface Properties and Chemistry
[0066] Most of large-sized tissues and organs with distinct
three-dimensional form will require support for their formation
from cells. For example, stem cells reside in a specialized
microenvironment called stem cell niche which provides the stem
cells with extracellular stimuli to allow their survival and
identity. This niche is a key regulator to the stem cell behavior
because it ensures a quiescent and low metabolic environment to
prevent exhaustion. It is believed that microenvironmental
properties of the niche provide a good balance between the ability
of stem cells to renew themselves and the ability to differentiate
into mature cells so that continuous tissue regeneration occurs. A
major part of the cell niche is the ECM (extracellular matrix)
which possesses the specific mechanical, biochemical, and
biophysical properties for tissues and controls the overall cell
behavior. The composition of the ECM provides full support to the
niche through its physical and structural properties. The major
function of the artificial support is similar to that of the
natural ECM that assists in proliferation, differentiation, and
biosynthesis of cells. For this reason, it is desired that the
surface of the culture unit not only comprises biochemical (e.g.
functionality-inducing) stimuli but also has definite physical
properties such as definite mechanical properties including e.g.
surface stiffness/flexibility.
[0067] The physical properties may be, at least partly, provided by
the elastomer material of the substrate or scaffold in that it
provides a form of stiffness or flexibility. According to the IUPAC
an "elastomer" is a polymer or polymeric material that displays
rubber-like elasticity. Elastomer materials are therefore also
referred to as rubber materials. An elastomer material generally is
a polymeric material with viscoelasticity (i.e., both viscosity and
elasticity) and generally has a low Young's modulus and high
failure strain compared with other materials. The elastomers
generally are polymer materials, many of which have polymer
backbones held together by intermolecular forces such as provided
by crosslinks.
[0068] The elastomer to be used with the invention may be any type
of elastomer. For example, the elastomer may be a homopolymer, a
copolymer, a block copolymer, a terpolymer, a block terpolymer and
so on or a mixture of two or more of these. A particularly suitable
elastomer may be selected from polyenes such as polybutadiene
and/or silicones (polysiloxanes). A suitable polysiloxane comprises
or consists of polydimethylsiloxane (PDMS). The polysiloxane may
comprise a crosslinked siloxane backbone. The crosslinking may have
been obtained by hydrosilylation reactions as known in the art.
between vinyl moieties and silylhydride moyeties of polysiloxane
precursors making use of appropriate hydrosylilation catalysts e.g.
Pt-catalysts.
[0069] In an embodiment, the elastomer material comprises or
consists of a polysiloxane and/or polydiene material. Preferably
the polysiloxane comprises or consists polydimethylsiloxane (PDMS).
A preferred polydiene material is polybutadiene.
[0070] Many elastomers are known in the art. However, the elastomer
materials of the invention are modified in that they have the
stimuli coupled to them (i.e. the elastomer material bears the
stimuli). At least part of such coupled stimuli should be available
at the elastomer surface. In this way they are available for
providing the desired stimulus to cells during cell culturing.
Coupling in this respect may mean any type of chemical or physical
attachment, but preferably includes or consists of covalent bonding
via connective chemical groups. Examples of such modifications and
couplings will be described hereinbelow.
[0071] Many of the stimuli coupled to the elastomer are organic
molecules and, are also proteins. As known many proteins have
carboxylic acid or amine bearing aminoacids at their outer
periphery which may be used for chemical coupling or labelling. Any
type or way of coupling that preserves the stimulus function of
such organic molecules or proteins when coupled to the elastomer
material can in principle be used. In the field of chemistry known
as bioconjugation, many different coupling reactions leading to a
variety of coupling groups are known. Confer for example "Surface
modification Chemistries of Materials used in diagnostic platforms
with biomolecules", by M. D. Sonawane and S. B. Nimse, and
published in Journal of Chemistry, vol. 16, page 1 e.v. and Thermo
Scientific Crosslinking Handbook; easy molecular bonding
crosslinking technology, Reactivity chemistries, applications and
structure references, 2012, available via download on the internet
page:
http://tools.thermofisher.com/content/sfs/brochures/1602163-Crosslinking--
Reagents-Handbook.pdf. Especially the latter reference lists
various chemistries that can be used for coupling for the current
invention. In particular the paragraph on protein immobilization
onto solid supports provides guidance as to how to couple proteins
to sold surfaces having carboxy and amine modified elastomer
polymers. Examples of suitable coupling chemistry would be based on
N-hydroxysuccinimide (NHS) esters as activated carboxylate
functional groups for coupling to e.g. primary amines. Often
carbodiimides such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) or N,N'-Dicyclohexylcarbodiimide (DCC) are used to in an
intermediate step activate a carboxylic acid group for reaction
with NHS.
[0072] To allow for such coupling to take place a precursor
elastomer material may be used which has been functionalized with
reactive groups for coupling of the stimuli.using chemistries as
e.g. mentioned herein above. Example reactive groups can be
selected from carboxylic acid or conjugate base, carboxylate, and
amine groups, but others may also be used. Thus for example
carboxylic acid groups of the elastomer material precursor may be
used to couple to amine groups of the stimuli or vice versa. The
functionalization can be achieved in different ways. While a first
group of elastomer precursor materials after their formation
(polymerization) may have functional groups available for further
reaction (for example polybutadiene) another group requires
explicit functionalization (for example many polysiloxanes such as
PDMS or the like). Functionalization can be achieved in different
ways. For example, a precursor elastomer material such as PDMS may
be surface treated (after its polymerization) using various kinds
of treatments, such as for example oxidation via plasma etc. These
are techniques well known in the art. In a further method,
appropriate chemical groups are incorporated in the precursor
elastomer material during its formation by polymerization. Such
methods are for example described herein and used for manufacture
of the experimental samples described herein.
[0073] In an embodiment, the elastomer material comprises or
consists of a polysiloxane and/or polydiene material wherein the
one or more stimuli are coupled to the surface via carboxyl groups.
Advantageously, coupling the one or more stimuli to carboxyl groups
sticking out of surface material such as PDMS or Lucas rubber
mimics in vivo situation for example for cell receptor ligands of
members of the TGF-.beta. family, ligands of the Hedgehog and Notch
pathways, and ligands of integrin-induced cell signaling. As a
further advantage, variations in substrate stiffness can be easily
obtained with both rubber and PDMS.
[0074] According to a preferred embodiment, the surface comprises
or is composed of a material for binding said one or more stimuli
such as cell receptor ligand, wherein the material corresponds to a
material for binding to a cell culturing protein as described in
international patent application PCT/EP2018/068318, titled "Cell
culturing materials", published as PCT patent application WO
2019/015988, having a filing date of 6 Jul. 2018 and claiming
priority of EP application number 17181708.5 filed on 18 Jul. 2017,
which disclosure is herein incorporated by reference in its
entirety. Particular reference is made to the surface materials,
and surface chemistry that render the surface materials suitable
for binding a cell culturing protein. The therein disclosed
material is capable of binding the herein described one or more
stimuli in place of the cell culturing protein described in the
afore-said international patent application.
[0075] According to a further preferred embodiment, the scaffold,
as disclosed herein, is a scaffold obtained by functionalizing the
polymeric surface before coupling the functionality-inducing one or
more stimuli. Functionalization is preferably carried out using
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC),
followed by reaction with N-hydroxysuccinimide (NHS), as disclosed
herein or in PCT patent application WO 2019/015988.
[0076] EDC/NHS are initiators (catalysts) which will be washed out
after coupling the functional protein. These initiators are
standard. They are not detectable after functionalization.
[0077] For the purposes of the present description and experiments,
the material in particular contains a bulk-modified elastomer
comprising a plurality of fatty acid moieties covalently bound to
the elastomer bulk, wherein the carboxylic acid (carboxylic) groups
of said moieties are available to provide binding to the one or
more stimuli. Such a material can advantageously be manufactured in
a few steps. In some embodiments, a single step suffices, for
example when the manufacturing is carried out by combining the
elastomer and the fatty acid in a coating process (e.g., spin
coating, dip coating, spray coating, dispensing) or an injection
molding process in which the cross-linking of the elastomer with
the fatty acid carbon-carbon double bond can be achieved without
significant epoxidation of the fatty acid carbon-carbon double bond
due to the limited exposure to ambient oxygen in the coating or
injection molding process. Thereby, a material is provided in which
the elastomer is bulk modified with fatty acid moieties in which
the carboxylic acid groups of the fatty acid are available to bind
to a biocompatible material such as the one or more stimuli
described herein.
[0078] Moreover, by controlling curing and/or properties of the
mold in which the material is formed, such carboxylic acid groups
can predominantly present on an outer surface of the material,
thereby providing a substantially homogeneous distribution of
carboxylic acid groups on such outer surface, which makes the
material particularly suitable for use as a membrane material for a
fluidic device, as each cross-section of the material will exhibit
the same surface properties, in contrast to membrane materials onto
which an anchor for the biocompatible material needs to be grafted
or otherwise formed as with known techniques. In addition, because
typically only a fraction of the elastomer carbon-carbon double
bonds is consumed in such a cross-linking reaction, the material
retains the elastomeric properties of the elastomer, which adds to
the suitability of the material for use in fluidic devices and
facilitates the slicing or otherwise cutting of the material for
investigative or other purposes.
[0079] Preferably, each of the fatty acid moieties is covalently
bound to the elastomer bulk through a cross-linking reaction
between a vinyl functional group or a hydride functional group of
the elastomer and an unsaturated carbon-carbon bond of an
unsaturated fatty acid to ensure that the number of carboxylic acid
groups available for binding to the biocompatible material can be
optimized.
[0080] The elastomer may be any suitable elastomer. For example,
the elastomer may be a homopolymer, a copolymer, a block copolymer,
a terpolymer, a block terpolymer and so on. A particularly suitable
elastomer may be selected from polyenes such as polybutadiene or
silicones (polysiloxanes). Such silicones may comprise a PDMS
backbone including vinyl moieties to facilitate cross-linking
through Pt-catalyzed addition reactions, e.g. with (poly methyl)
hydrogen siloxanes. Alternatively, a silicone backbone such as a
(poly methyl) hydrogen siloxane backbone may be crosslinked with
rubber-like polymers such as polybutadiene and polyisoprene, with
the unsaturated fatty acid being incorporated in such a crosslinked
product.
[0081] Regarding to unsaturated fatty acid, any suitable
unsaturated fatty acid may be used for the purpose of cross-linking
it with the elastomer. For example, the unsaturated fatty acid may
be selected from myristoleic acid, palmitoleic acid, sapienic acid,
oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoeladic
acid, a-linolenic acid, arachidonic acid, eicospaentaenoic acid,
erucic acid and docosahexaenoic acid. Linoleic acid is specifically
mentioned.
[0082] The elastomer and the unsaturated fatty acid may be mixed in
any suitable ratio in a composition to form the bulk-modified
elastomer. Preferably, unsaturated fatty acid is present in such a
composition in a range of 0.05-35% by weight of the elastomer such
that upon bulk modification of the elastomer the bulk-modified
elastomer retains its flexibility to the remaining presence of
carbon-carbon double bonds in the elastomer bulk.
[0083] It will be clear that other ways of obtaining elastomer
materials with stimuli coupled to them may be used to good effect
in achieving the cell functionalization as described herein
before.
[0084] If a patterning of stimuli as described above is desired, a
fluidic device with more than one membrane may be used, wherein
each membrane can be advantageously manufactured as described
above. The membranes may be stacked so that cell can be cultivated
in front of each other. In this way, for example liver tissue and
pancreas tissue can be grown in front of each other. The
consumption of glucose in the nutrition for both organs and the
production of insulin by the pancreas could be studied in a
co-working organ on chip system.
Microfluidic Devices
[0085] Control of cellular behavior such as stem cell culture and
differentiation require precise control of multiple stimuli in the
cell culture microenvironment, which regulate intracellular
signaling and ultimately cell phenotype. Microfluidic devices,
which can control multiple stimuli simultaneously over space and
time with high precision, provide an ideal and well-defined
platform for cultivating cells such as stem cells in a controlled
manner.
[0086] According to a preferred embodiment, the cell culturing
scaffold is part of a fluidic device module including at least one
flow channel extending over a membrane, the membrane comprising
said cell culturing scaffold. That is, the culture unit is a
microfluidic device. Such a fluidic device module may comprise a
first major surface comprising a first recessed structure defining
a first flow channel. Depending on the application, a second major
surface opposing the first major surface and comprising a second
recessed structure defining a second flow channel may be foreseen
with the membrane separating the first flow channel from the second
flow channel. This is particular useful in case the cell culture
shall be later used in the same device to monitor the cells'
response to a drug as described in the aforementioned international
patent application. In a preferred embodiment, the fluidic device
module is a monolithic fluidic device module, which has the
advantage that the device module can be manufactured in a small
number of processing steps, e.g. a single processing step, by
injection molding.
[0087] The membrane may comprise a plurality of holes or grooves
extending through the membrane. The holes or grooves have
preferably cell-size dimensions in order to retain the cells to be
cultured.
[0088] In an embodiment, the first flow channel and the second flow
channel are accessible through respective septa, such that the
separate flow channels may be individually accessed without
cross-contamination risk.
[0089] The fluidic device module may further comprise a pair of
cover plates (e.g. transparent glass sheets), wherein one cover
sheet comprises preferably two inlets and two outlets, to fluidly
seal the fluidic device module. The cover plates may be arranged
such that one of said cover plates covers the first major surface,
thereby sealing the first fluidic channel and the other of said
cover plates covers the second major surface, thereby sealing the
second fluidic channel. Such a fluidic device can be manufactured
in a straightforward manner, as the fluidic device module may be
formed in a small number of processing steps as previously
explained and is robust against leakage due to the flexible nature
of the fluidic device module. If the membrane is made of an
elastomer material, in particular a soft flexible elastomer
material, the cover plates advantageously forms together with the
membrane clamped between the cover plates a leakage tight fluidic
device module.
[0090] In a preferred embodiment, the membrane is capable of
adhering to the top and/or bottom cover plates. Consequently, after
clamping the membrane and the cover plates together, said parts
stay together due to the membrane's sticky properties. Thereby, it
is possible to handle the fluidic device module under/on the
microscope such as a confocal microscope after having the fluidic
device module removed from a fluidic system like Micronit or
Fluigent without risk that the cover plates, being in particular
made of a transparent material to facilitate microscopy, detach
from the membrane.
[0091] It is also envisaged that the membrane allows the top cover
plate to be easily removed, whereas it adheres to the bottom cover
plate so that the cell culture, organ, tissue or biopsy on the
membrane can be bended towards the bottom cover plate. Thereby, the
bottom cover plate remains in touch with lower side of the membrane
and allows the membrane to be positioned precisely in focus of a
confocal microscope.
[0092] In order to change culture conditions from the first culture
conditions to the second culture conditions, etc. at different
times, the cells grown on a first surface and/or membrane may be
detached and replaced by a second surface and/or membrane
comprising one or more second stimuli. Advantageously, a plurality
of different stimuli-precoated surfaces and/or membranes may be
prepared and provided in a kit format for this purpose.
[0093] For a detailed description of suitable manufacturing
methods, further suitable unsaturated fatty acids and elastomers,
particulars with regard to structural modifications of the membrane
and/or configurations of the fluidic device module, the reader is
referred to the above-cited international patent application.
Cell Types
[0094] The cell is not particularly limited and can be any cell or
cell line that is intended to acquire a particular cellular
behavior. According to a specific embodiment, the cells are
selected from the group consisting of mouse stem cells, human stem
cells, pluripotent stem cells, mesenchymal stem cells, embryonic
stem cells, adult stem cells and tumor cells.
[0095] Stem cells have enormous potential in numerous clinically
related applications including tissue engineering, organ
regeneration, cell-based therapies, disease models, drug response
testing, drug screening and development and a variety of healthcare
applications. The primary step in most applications involving stem
cells is to direct the differentiation of the cells to the desired
progeny. Naturally, there are two types of stem cells, the first
being embryonic stem cells (ESCs) and the second being adult stem
cells. ESCs are isolated from the inner cell mass of blastocysts
and have the pluripotency to differentiate into virtually all cell
lineages. Adult stem cells can be found in various tissues and can
differentiate to a limited number of cell types. In addition, there
are reprogrammed human-induced pluripotent stem cells (hiPSCs),
which can be directly obtained from adult cells by introduction of
specific genes. Because they can propagate indefinitely, as well as
give rise to every other cell type in the body, they represent a
single source of cells that could be used to replace those lost due
to damage or disease. In certain embodiments, the cells employed in
the present method do not require destruction of an embryo. In such
certain embodiments, embryonic stem cells are excluded from the
scope of the invention.
[0096] Tumor cells may be used for producing a cancer model system,
for the purposes of diagnosing or subtyping a cancer and/or testing
response of the cancer cells to drugs.
Pathway Analysis
[0097] According to a further preferred embodiment, the at least
one signaling pathway is selected from the group of: [0098] nuclear
receptors, like ER, AR, progesterone receptor; [0099] growth factor
pathways, like PI3K-FOXO, JAK-STAT3, MAPK-AP1 pathways; [0100]
immune pathways like STAT1/2 type I interferon (STAT1/2-1), STAT1/2
type II interferon (STAT1/2-2) pathways; [0101] developmental
pathways, like Hedgehog, Notch, TGF-.beta., and Wnt signaling
pathways; and [0102] the inflammatory pathway NFkB.
[0103] According to a further preferred embodiment, the activity of
the at least one first and/or second, etc., signaling pathway in
the cell sample is inferable by a method comprising: [0104]
receiving expression levels of three or more target genes of the at
least one signaling pathway in at least one cell contained in the
cell sample, [0105] determining an activity level of a
transcription factor (TF) element in the at least one cell, the TF
element controlling transcription of the three or more target
genes, the determining being based on evaluating a calibrated
mathematical pathway model relating expression levels of the three
or more target genes to the activity level of the at least one
signaling pathway, and [0106] inferring the activity of the at
least one signaling pathway in the at least one cell based on the
determined activity level of the TF element, wherein the calibrated
mathematical pathway model is preferably a centroid, linear or
Bayesian network model, based on conditional probabilities.
[0107] The term "activity of the at least one signaling pathway"
may refer to the activity of a signaling pathway associated
transcription factor (TF) element in the sample, the TF element
controlling transcription of target genes, in driving the target
genes to expression, i.e., the speed by which the target genes are
transcribed, e.g. in terms of high activity (i.e. high speed) or
low activity (i.e. low speed), or respective values, levels,
scores, dimensions or the like related to or indicative for such
activity (e.g. speed). Accordingly, for the purposes of the present
invention, the term "activity", as used herein, is also meant to
refer to said activity level. Both the activity and the activity
level can be determined by pathway analysis as described herein. As
described above, the activity may be identical to the activity
level. Such level can be directly used as an input for step
(d).
[0108] The pathway activity is determinable, e.g., in a sample
isolated from the cell culture, by pathway analysis as described
herein.
[0109] Pathway analysis enables quantitative measurement of signal
transduction pathway activity in cells present in tissue/cell
samples, based on inferring activity of a signal transduction
pathway from measurements of mRNA levels of the well-validated
direct target genes of the transcription factor associated with the
respective signaling pathway (see for example W Verhaegh, et al.,
Selection of personalized patient therapy through the use of
knowledge-based computational models that identify tumor-driving
signal transduction pathways. Cancer research 2014; 74(11):2936-45;
W Verhaegh, A van de Stolpe. Knowledge-based computational models.
Oncotarget 2014; 5(14):5196).
[0110] According to a preferred embodiment of the various aspects
of the present invention and the various embodiments thereof, as
disclosed herein, the determining of the activity of one or more
pathways, the combination of multiple pathway activities and
applications thereof is performed as described for example in the
following documents, each of which is hereby incorporated in its
entirety for the purposes of determining activity of the respective
signaling pathway: published international patent applications
WO2013011479 (titled "ASSESSMENT OF CELLULAR SIGNALING PATHWAY
ACTIVITY USING PROBABILISTIC MODELING OF TARGET GENE EXPRESSION"),
WO2014102668 (titled "ASSESSMENT OF CELLULAR SIGNALING PATHWAY
ACTIVITY USING LINEAR COMBINATION(S) OF TARGET GENE EXPRESSIONS"),
WO2015101635 (titled "ASSESSMENT OF THE PI3K CELLULAR SIGNALING
PATHWAY ACTIVITY USING MATHEMATICAL MODELLING OF TARGET GENE
EXPRESSION"), WO2016062891 (titled "ASSESSMENT OF TGF-(3 CELLULAR
SIGNALING PATHWAY ACTIVITY USING MATHEMATICAL MODELLING OF TARGET
GENE EXPRESSION"), WO2017029215 (titled "ASSESSMENT OF NFKB
CELLULAR SIGNALING PATHWAY ACTIVITY USING MATHEMATICAL MODELLING OF
TARGET GENE EXPRESSION"), WO2014174003 (titled "MEDICAL PROGNOSIS
AND PREDICTION OF TREATMENT RESPONSE USING MULTIPLE CELLULAR
SIGNALLING PATHWAY ACTIVITIES"), WO2016062892 (titled "MEDICAL
PROGNOSIS AND PREDICTION OF TREATMENT RESPONSE USING MULTIPLE
CELLULAR SIGNALING PATHWAY ACTIVITIES"), WO2016062893 (titled
"MEDICAL PROGNOSIS AND PREDICTION OF TREATMENT RESPONSE USING
MULTIPLE CELLULAR SIGNALING PATHWAY ACTIVITIES") and in the patent
applications EP16200697.7 (filed on Nov. 25, 2016; titled "Method
to distinguish tumor suppressive FOXO activity from oxidative
stress"), EP17194288.1 (filed on Oct. 2, 2017; titled "Assessment
of Notch cellular signaling pathway activity using mathematical
modelling of target gene expression"), EP17194291.5 (filed on Oct
2, 2017; titled "Assessment of JAK-STAT1/2 cellular signaling
pathway activity using mathematical modelling of target gene
expression"), EP17194293.1 (filed on Oct 2, 2017; titled
"Assessment of JAK-STAT3 cellular signaling pathway activity using
mathematical modelling of target gene expression") and EP17209053.2
(filed on Dec. 20, 2017, titled "Assessment of MAPK-AP1 cellular
signaling pathway activity using mathematical modelling of target
gene expression"). The models have been biologically validated for
ER, AR, PI3K-FOXO, HH, Notch, TGF-.beta., Wnt, NFkB, JAK-STAT1/2,
JAK-STAT3 and MAPK-AP1 pathways on several cell types. It is noted
that the mathematical models employed in the patent applications
that are not yet published as well as the calibration and use of
these models in these applications generally correspond to the
models, calibration and use disclosed in the already published
patent applications.
[0111] To facilitate rapid identification of references, the
above-mentioned references have been assigned to each signaling
pathway of interest here and exemplarily corresponding target genes
suitable for determination of the signaling pathway's activity have
been indicated. In this respect, particular reference is also made
to the sequence listings for the target genes provided with the
above-mentioned references. [0112] Wnt: KIAA1199, AXIN2, RNF43,
TBX3, TDGF1, SOX9, ASCL2, IL8, SP5, ZNRF3, KLF6, CCND1, DEFA6 and
FZD7 (WO 2013/011479, WO 2014/102668, WO 2014/174003); ADRA2C,
ASCL2, AXIN2, BMP7, CCND1, CD44, COL18A1, DEFA6, DKK1, EPHB2,
EPHB3, FAT1, FZD7, GLUL, HNF1A, CXCL8 (previously known as IL8),
CEMIP (previously known as KIAA1 199), KLF6, LECT2, LEF1, LGRS,
MYC, NKD1, OAT, PPARG, REGIB, RNF43, SLC1A2, SOX9, SP5, TBX3,
TCF7L2, TDGF1, and ZNRF3 (WO 2016/062892, WO 2016/062893); [0113]
HH: GLI1, PTCH1, PTCH2, IGFBP6, SPP1, CCND2, FST, FOXL1, CFLAR,
TSC22D1, RAB34, S100A9, S100A7, MYCN, FOXM1, GLI3, TCEA2, FYN and
CTSL1 (WO 2013/011479, WO 2014/102668, WO 2014/174003); GLI1,
PTCH1, PTCH2, HHIP, SPP1, TSC22D1, CCND2, HI 9, IGFBP6, TOM1, JUP,
FOXA2, MYCN, NKX2-2, NKX2-8, RAB34, MIF, GLI3, FST, BCL2, CTSL1,
TCEA2, MYLK, FYN, PITRM1, CFLAR, IL1R2, S100A7, S100A9, CCND1,
JAG2, FOXMl, FOXF1, and FOXL1 (WO 2016/062892, WO 2016/062893);
[0114] AR: KLK2, PMEPA1, TMPRSS2, NKX3 1, ABCC4, KLK3, FKBP5, ELL2,
UGT2B15, DHCR24, PPAP2A, NDRG1, LRIG1, CREB3L4, LCP1, GUCY1A3, AR
and EAF2 (WO 2013/011479, WO 2014/102668); KLK2, PMEPA1, TMPRSS2,
NKX3 1, ABCC4, KLK3, FKBP5, ELL2, UGT2B15, DHCR24, PPAP2A, NDRG1,
LRIG1, CREB3L4, LCP1, GUCY1A3, AR, and EAF2 (WO 2014/174003);
[0115] ER: CDH26, SGK3, PGR, GREB1, CA12, XBP1, CELSR2, WISP2,
DSCAM, ERBB2, CTSD, TFF1 and NRIP1 (WO 2013/011479, WO
2014/102668); GREB1, PGR, XBP1, CA12, SOD1, CTSD, IGFBP4, TFF1,
SGK3, NRIP1, CELSR2, WISP2, and AP1B1 (WO 2014/174003); AP1B1,
ATP5J, COL18A1, COX7A2L, CTSD, DSCAM, EBAG9, ESR1, HSPB1, KRT19,
NDUFV3, NRIPI, PGR, PISD, PRDM15, PTMA, RARA, SOD1, TFF1, TRIM25,
XBP1, GREB1, IGFBP4, MYC, SGK3, WISP2, ERBB2, CA12, CDH26, and
CELSR2 (WO 2016/062892, WO 2016/062893); [0116] PI3K-FOXO: AGRP,
BCL2L11, BCL6, BNIP3, BTG1, CAT, CAV1, CCND1, CCND2, CCNG2, CDK 1A,
CDK 1B, ESR1, FASLG, FBX032, GADD45A, INSR, MXI1, NOS3, PCK1, POMC,
PPARGCIA, PRDX3, RBL2, SOD2 and TNFSF10 (WO 2015/101635); ATP8A1,
BCL2L11, BNIP3, BTG1, C1Oorf1O, CAT, CBLB, CCND1, CCND2, CDKNIB,
DDB1, DYRK2, ERBB3, EREG, ESR1, EXT1, FASLG, FGFR2, GADD45A, IGF1R,
IGFBP1, IGFBP3, INSR, LGMN, MXI1, PPM1D, SEMA3C, SEPP1, SESN1,
SLC5A3, SMAD4, SOD2, TLE4, and TNFSF10 (WO 2016/062892, WO
2016/062893); SOD2, BNIP3, MXI1, PCK1, PPARGC1A and CAT
(EP16200697.7, supra); [0117] TGF-.beta.: ANGPTL4, CDC42EP3,
CDKNIA, CDKN2B, CTGF, GADD45A, GADD45B, HMGA2, ID1, IL11, SERPINE1,
INPP5D, JUNB, MMP2, MMP9, NKX2-5, OVOL1, PDGFB, PTHLH, SGK1, SKIL,
SMAD4, SMADS, SMAD6, SMAD7, SNAIl, SNAI2, TIMP1 and VEGFA (WO
2016/062891, WO 2016/062893); [0118] NFkB: BCL2L1, BIRC3, CCL2,
CCL3, CCL4, CCLS, CCL20, CCL22, CX3CL1, CXCL1, CXCL2, CXCL3, ICAM1,
IL1B, IL6, IL8, IRF1, MMP9, NFKB2, NFKBIA, NFKB IE, PTGS2, SELE,
STATSA, TNF, TNFAIP2, TNIP1, TRAF1 and VCAM1 (WO 2017/029215);
[0119] Notch: CD28, CD44, DLGAP5, DTX1, EPHB3, FABP7, GFAP, GIMAP5,
HES1, HES4, HES5, HES7, HEY1, HEY2, HEYL, KLF5, MYC, NFKB2, NOX1,
NRARP, PBX1, PIN1, PLXND1, PTCRA, SOX9 and TNC (EP 17194288.1,
supra); [0120] JAK-STAT1/2: BID, GNAZ, IRF1, IRF7, IRF8, IRF9,
LGALS1, NCF4, NFAM1, OAS1, PDCD1, RAB36, RBX1, RFPL3, SAMM50,
SMARCB1, SSTR3, ST13, STAT1, TRMT1, UFD1L, USP18, and ZNRF3,
preferably, from the group consisting of: IRF1, IRF7, IRF8, IRF9,
OAS1, PDCD1, ST13, STAT1 and USP18 (EP17194291.5, supra); [0121]
JAK-STAT3: AKT1, BCL2, BCL2L1, BIRC5, CCND1, CD274, CDKN1A, CRP,
FGF2, FOS, FSCN1, FSCN2, FSCN3, HIF1A, HSP90AA1, HSP90AB1, HSP90B1,
HSPA1A, HSPA1B, ICAM1, IFNG, IL 10, JunB, MCL1, MMP1, MMP3, MMP9,
MUC1, MYC, NOS2, POU2F1, PTGS2, SAA1, STAT1, TIMP1, TNFRSF1B,
TWIST1, VIM and ZEB1 (EP17194293.1, supra); [0122] MAPK-AP1:
BCL2L11, CCND1, DDIT3, DNMT1, EGFR, ENPP2, EZR, FASLG, FIGF, GLRX,
IL2, IVL, LOR, MMP1, MMP3, MMP9, SERPINE1, PLAU, PLAUR, PTGS2,
SNCG, TIMP1, TP53 and VIM (EP17209053.2, supra).
[0123] Common to the pathway analysis methods for determining the
activities of the different signaling pathways as disclosed herein
is a concept, which is preferably applied herein for the purposes
of the present invention, wherein the activity of a signaling
pathway in a cell present in a sample is determinable by receiving
expression levels of one or more, preferably three or more, target
genes of the signaling pathway, determining an activity level of a
signaling pathway associated transcription factor (TF) element in
the sample, the TF element controlling transcription of the three
or more target genes, the determining being based on evaluating a
calibrated mathematical pathway model relating expression levels of
the three or more target genes to the activity level of the
signaling pathway, and optionally inferring the activity of the
signaling pathway in the cell based on the determined activity
level of the signaling pathway associated TF element in the
sample.
[0124] The term "transcription factor element" (TF element), as
used herein, preferably refers to an intermediate or precursor
protein or protein complex of the active transcription factor, or
an active transcription factor protein or protein complex which
controls the specified target gene expression. For example, the
protein complex may contain at least the intracellular domain of
one of the respective signaling pathway proteins, with one or more
co-factors, thereby controlling transcription of target genes.
Preferably, the term refers to either a protein or protein complex
transcriptional factor triggered by the cleavage of one of the
respective signaling pathway proteins resulting in an intracellular
domain.
[0125] As indicated above, the term "activity level" of a TF
element, as used herein, denotes the level of activity of the TF
element regarding transcription of its target genes.
[0126] The term "target gene", as used herein, means a gene whose
transcription is directly or indirectly controlled by a respective
transcription factor element. The "target gene" may be a "direct
target gene" and/or an "indirect target gene" (as described
herein).
[0127] Particular reference is further made to the definitions of
the respective pathways, the respective transcription factor
elements, the (sets of) target genes, the calibration methodology
(using as calibrated mathematical pathway model a model that is
calibrated using a ground truth dataset including samples in which
transcription of several, e.g. three or more, target genes of the
respective cellular signaling pathway is induced by the respective
TF element(s) and samples in which transcription of the several,
e.g. the three or more, target genes is not induced by the
respective TF element(s)), provided in the aforementioned patent
applications.
[0128] The calibrated mathematical pathway model may be a
probabilistic model, preferably a Bayesian network model, based on
conditional probabilities relating the activity level of the
signaling pathway associated TF element and the expression levels
of the three or more target genes, or the calibrated mathematical
pathway model may be based on one or more linear combination(s) of
the expression levels of the three or more target genes. For the
purposes of the present invention, the calibrated mathematical
pathway model is preferably a centroid or a linear model, or a
Bayesian network model based on conditional probabilities.
[0129] In particular, the determination of the expression level and
optionally the inferring of the activity of a signaling pathway in
the cell sample may be performed, for example, by inter alfa (i)
evaluating a portion of a calibrated probabilistic pathway model,
preferably a Bayesian network, representing the cellular signaling
pathways for a set of inputs including the expression levels of the
three or more target genes of the cellular signaling pathway
measured in the cell sample, (ii) estimating an activity level in
the cell sample of a signaling pathway associated transcription
factor (TF) element, the signaling pathway associated TF element
controlling transcription of the three or more target genes of the
cellular signaling pathway, the estimating being based on
conditional probabilities relating the activity level of the
signaling pathway associated TF element and the expression levels
of the three or more target genes of the cellular signaling pathway
measured in the cell sample, and optionally (iii) inferring the
activity of the cellular signaling pathway based on the estimated
activity level of the signaling pathway associated TF element in
the cell sample. This is described in detail in the published
international patent application WO 2013/011479 A2 ("Assessment of
cellular signaling pathway activity using probabilistic modeling of
target gene expression"), the contents of which are herewith
incorporated in their entirety.
[0130] In an exemplary alternative, the determination of the
expression level and optionally the inferring of the activity of a
cellular signaling pathway may be performed by inter alia (i)
determining an activity level of a signaling pathway associated
transcription factor (TF) element in the cell sample, the signaling
pathway associated TF element controlling transcription of the
three or more target genes of the cellular signaling pathway, the
determining being based on evaluating a calibrated mathematical
pathway model relating expression levels of the three or more
target genes of the cellular signaling pathway to the activity
level of the signaling pathway associated TF element, the
mathematical pathway model being based on one or more linear
combination(s) of expression levels of the three or more target
genes, and optionally (ii) inferring the activity of the cellular
signaling pathway in the cells of the cell culture based on the
determined activity level of the signaling pathway associated TF
element in the cell sample. This is described in detail in the
published international patent application WO 2014/102668 A2
("Assessment of cellular signaling pathway activity using linear
combination(s) of target gene expressions").
[0131] Further details regarding the inferring of cellular
signaling pathway activity using mathematical modeling of target
gene expression can be found in Verhaegh W. et al., "Selection of
personalized patient therapy through the use of knowledge-based
computational models that identify tumor-driving signal
transduction pathways", Cancer Research, Vol. 74, No. 11, 2014,
pages 2936 to 2945.
[0132] In an embodiment the signaling pathway measurements are
performed using qPCR, multiple qPCR, multiplexed qPCR, ddPCR,
RNAseq, RNA expression array or mass spectrometry. For example, a
gene expression microarray data, e.g. Affymetrix microarray, or RNA
sequencing methods, like an Illumina sequencer, can be used.
Storage Medium, Computer Program and System
[0133] In accordance with a fourth aspect of the present invention,
a non-transitory storage medium stores instruction that are
executable by a digital processing device to perform the method
according to the third aspect of the invention, and the various
embodiments thereof. The non-transitory storage medium may be a
computer-readable storage medium, such as a hard drive or other
magnetic storage medium, an optical disk or other optical storage
medium, a random access memory (RAM), read only memory (ROM), flash
memory, or other electronic storage medium, a network server, or so
forth. The digital processing device may be a handheld device
(e.g., a personal data assistant or smartphone), a notebook
computer, a desktop computer, a tablet computer or device, a remote
network server, or so forth.
[0134] In accordance with a fifth aspect of the present invention,
a computer program comprises program code means for causing a
digital processing device to perform the method according to the
third aspect of the invention, and the various embodiments thereof,
when the computer program is run on the digital processing device.
The digital processing device may be a handheld device (e.g., a
personal data assistant or smartphone), a notebook computer, a
desktop computer, a tablet computer or device, a remote network
server, and so forth.
[0135] In accordance with a sixth aspect of the present invention,
a system for performing the method of the invention comprises
[0136] (a) a culture unit or a scaffold, preferably as defined
herein, [0137] (b) a sensor unit for inferring an activity of at
least one first signaling pathway; [0138] (c) a controller that is
configured to compare the inferred activity with a predetermined
activity of the at least one first signaling pathway, the
predetermined activity being selected so to control cell behavior
in a first predetermined manner, i.e. to controllably steer the
cells into a predetermined direction of cell functionality, and
optionally [0139] (d) a non-transitory storage medium of the
invention or a computer program of the invention.
[0140] According to a preferred embodiment, the controller is
further configured to determine culture conditions based on the
comparison of the inferred activity with the selected activity, the
culture conditions being optionally determined so as to (ii) reduce
deviation of the inferred activity from the selected activity or
(ii) control activity of at least one second signaling pathway, the
activity of the at least one second signaling pathway.
[0141] The culture unit is preferably constituted as described
herein in the context of the various aspects of the invention, as
disclosed herein. The sensor unit is adapted to infer the activity
of the at least one first signaling pathway. To this end, the
sensor unit may be equipped with one or more components or means
for measuring the expression levels of the target genes such as a
DNA array chip, an oligonucleotide array chip, a protein array
chip, an antibody, a plurality of probes, for example, labeled
probes, a set of RNA reverse-transcriptase sequencing components,
and/or RNA or DNA, including cDNA or amplification primers.
Preferably, the sensor unit is adapted to perform one of qPCR,
multiple qPCR, multiplexed qPCR, ddPCR, RNAseq, RNA expression
array and mass spectrometry.
[0142] In an embodiment, the sensor unit uses a set of labeled
probes directed to a portion of an mRNA or cDNA sequence of the
target genes as described herein. In an embodiment, the sensor unit
uses a set of primers and probes directed to a portion of an mRNA
or cDNA sequence of the target genes. In an embodiment, the labeled
probes are contained in a standardized 96-well plate. In an
embodiment, the sensor unit further uses primers or probes directed
to a set of reference genes. Such reference genes can be, for
example, constitutively expressed genes useful in normalizing or
standardizing expression levels of the target gene expression
levels described herein.
[0143] The sensor unit may further include a processor to compute
results corresponding to the inferred activity of the at least one
first signaling pathway.
Uses
[0144] The various aspects of the present invention are
particularly useful for: [0145] determining, predicting or
controlling that a cell culture acquires a particular cellular
behavior; [0146] determining, predicting or controlling that a cell
culture differentiated to or in the direction of the desired
progeny; [0147] determining, predicting or controlling that a cell
culture acquires a particular function; [0148] determining,
predicting or controlling cell culture response to particular
stimuli; producing a cell culture, tissue or organ in a controlled
and robust manner; [0149] predicting, monitoring or determining
response to therapy using the cell culture, tissue or organ
produced by the method of the present invention; [0150] predicting,
monitoring or determining effectiveness of therapy using the cell
culture, tissue or organ produced by the method of the present
invention; [0151] producing tumor model systems; [0152] diagnosing
or subtyping a disease, in particular tumor disease; [0153]
producing cells suited for regenerative medicine purposes;
[0154] producing series of cells that are very well defined in a
quantitative manner with respect to their cellular state, e.g.
differentiation state, to enable reproducibility subsequent
experimental use, e.g. drug development or regenerative
medicine.
Exemplification of Differentiation of Stem Cells to Intestinal Stem
Cells
[0155] Stem cells (e.g. iPS or HES cells) can be differentiated in
standard culture to intestinal stem cells according to a protocol
described by Loh et al (supra).
[0156] Supplying the ligands (including those that cannot be added
to the medium like Notch ligands) to the cell culture in a
controlled manner in accordance with an embodiment of the present
invention to activate the respective signaling pathways that are
assumed to become active during various differentiation stages (cf.
FIG. 1, pathway analysis of a GEO dataset, differentiation to
hindgut; A. pathway activities measured per differentiation step;
B. overview picture shows sequential differentiation steps;
signaling pathway activities are indicated as 1og2 odds values per
individual sample) improves the outcome of the differentiation
experiment and will make such experiments more reproducible across
labs and time and render the method in particular useful for
standardization purposes of the experiment.
[0157] An exemplary experiment in accordance with the present
invention is carried out as follows. The experiment is designed
that the cells go through several differentiation steps, first to
obtain anterior primitive streak cells, subsequently definitive
endoderm, and then mid/hindgut cells phenotype.
[0158] Stem cells are cultured feeder-free at the required density
in defined serum-free CDM2 basal medium on tissue culture plastic.
Subsequently for the differentiation experiment they are passaged
and seeded on the herein described surface material that is
functionalized with a 50/50 mixture (10 microgram/ml) of Activin,
an appropriate Wnt ligand (e.g. Wnt 3A), in the presence of an mTOR
inhibitor in the CDM2 medium, e.g. LDN-193189/DM3189, (50 nM), for
24 hours to obtain anterior primitive streak cells. Subsequently,
cells are detached from the surface and pathway analysis is
performed on a cell sample for all mentioned signal transduction
pathways, to ascertain that the cells reached the required
differentiation step to a required quantitative extent. When this
pathway analysis produces satisfactory results, this enables a
positive decision to continue with the experiment. Cells are
subsequently seeded onto a next substrate functionalized with
Activin in the presence of the BMP-inhibitor LDN-193189/DM3189 (250
nM) in CDM2 for 48 hours to obtain definitive endoderm. Cells are
detached again, differentiation status again measured in a
quantitative manner using pathway analysis for all mentioned signal
transduction pathways. If confirmative, cells are reseeded and
cultured on a herein described surface material functionalized with
a 15/15/35/35 mixture (10 microgram/ml) of BMP4, FGF2 and high Wnt
ligand (Wnt 3A) and Notch ligand (e.g. Delta) for 4 days in CDM2
medium containing retinoic acid (2 .mu.M, Sigma) and BMP-inhibitor
DM3189 (250 nM), optionally with the differentiating agent methoxy
substituted alkylglycerol-1-O-(2-methoxy) hexadecyl glycerol (MHG)
added, for 4 days. Media is refreshed every 24 hours. At the end,
cells are detached form the surface and pathway analysis can be
performed once more to ascertain that the required final
differentiation step has been obtained and to quantify the final
result (e.g. active Wnt and Notch pathway).
Exemplification of Functionalizing a Surface with a Cell Receptor
Ligand
[0159] An experiment designed as proof of principle for coupling a
cell receptor ligand to a functionalized surface is described using
TGF-.beta. as a representative example. The reason TGF-.beta. was
chosen is that the effect of TGF-.beta. can be shown by adding
TGF-.beta. to the medium as well, enabling performing a control
experiment for the effect of TGF-.beta. on the used cultured cells.
While the described method provides a large advantage over
currently used culture methods when coating the functionalized
material with a Hedgehog (HH) or Notch ligand, a control experiment
with these ligands would not have been possible since these ligands
preferably or only act when attached to a surface. However, when
coating of the functionalized surface with TGF-.beta. successfully
leads to induction of TGF-.beta. pathway activity in attached
cells, it can be safely concluded that coating with other protein
ligands will be similarly effective. TGFb1 was commercially bought
from STEMCELL TECHNOLOGIES INC. The TGF-.beta. coating experiment
was performed as follows: WPMY1 fibroblast cells were seeded and
cultured (DMEM medium with 10% FBS) on functionalized surface
(SIL-IM-F 0.5%, silicone substrate containing linoleic acid as
described herein and in international patent application
PCT/EP2018/068318 referred to above), which was according to
described protocol coated with TGFb1 by incubation with 10 ug TGFb1
(SIL-IM-F 0.5%-TGFb1), or as control non functionalized (SIL-IM-NF)
surface, similarly incubated with TGFb1 (SIL-IM-NF 0.5%). In
another control situation, WPMY1 cells were cultured on standard
tissue culture plastic and 1 ng/ml TGFb1 was added to the medium.
Cells were cultured for 48 hours, released from the surface and
signaling pathway analysis was performed on RNA isolated from the
cells as described herein and the patent application referred to
above in the context of determining activity of signaling pathways.
Cells attached and proliferated on all surfaces. A response to
TGF-.beta. was measured as a higher TGF-.beta. pathway score in the
standard culture (on culture plastic) compared to unstimulated
WPMY1 cells (52% higher), serving as control for responsiveness to
stimulation with TGFb1 in these cells (Table 1). TGFb1 coating of
functionalized surface resulted in 103% increase in TGF-.beta.
pathway activity; TGFb1 coating of non-functionalized surface
resulted in only 67% increase in TGF-(3 activity (cf. Table 1).
TABLE-US-00001 TABLE 1 Comparison of activation of TGF-.beta.
pathway by spiking TGFb1 or coating a non-functionalized and
functionalized surface with TGFb1. TGFB response With vs without
TGFb1 % increase control (medium spiked) 52% SIL-IM-NF
(non-functionalized surface; coated) 67% SIL-IM-F 0.5%
(functionalized; coated) 103%
[0160] This shows that the functionalized surface when coated with
TGFb1 was more effective in stimulating the TGF-.beta. pathway in
WPMY1 cells that are responsive to the TGF-.beta.ligand. In
agreement with this, the cell morphology changed compared to cells
on the non-TGFb1-coated surface (cf. FIG. 2).
[0161] In an independent cell culture experiment the tissue
culture-treated plastic surface, non-functionalized and
functionalized surface were incubated with different concentrations
of TGFb1, 0 ng/ml, 10 ng/ml and 1000 ng/ml (FIG. 3, indicated as
respectively NO, 10 ng and 1 microgram; on the y-axis is the
TGF-.beta. pathway activity score), and TGF-.beta. pathway activity
was analyzed. TGF-.beta. pathway activity increased with the
concentration of TGFb1 with which the functionalized surface had
been incubated to couple the TGFb1 to the COOH groups on the
functionalized surface. The increase was comparable to the effect
seen when cells were treated with 1 ng/ml TGFb1 in the culture
medium, when cultured on tissue culture-treated plastic. In
contrast to the non-functionalized surface a similar incubation
with TGFb1 did not increase TGF-.beta. pathway activity (FIG.
3).
Exemplification of Functionalizing a Surface with Another Cell
Receptor Ligand
[0162] Sample preparation: As described in PCT patent application
WO 2019/015988, the carboxy groups of the polymeric surface may be
reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC, compound II) followed by a reaction with
N-hydroxysuccinimide (NHS, compound IV), which can react with amine
groups of, e.g. a ligand to covalently bind the ligand to the
carboxy groups of the polymeric surface. In this manner, the ligand
can form a scaffold, as disclosed herein. Such a scaffold can be
formed with high uniformity in terms of coverage of the surface of
the polymeric surface, e.g. the surface of a membrane of a fluidic
device module due to the high density homogeneous distribution of
the carboxy groups across such a surface.
[0163] Accordingly, in this protocol, before ligand coupling to
modified silicone samples was done, a surface treatment of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC)/N-hydroxysuccinimide (NHS) was performed combining 0.4M EDC
(Sigma-Aldrich) with 0.1M NHS (Sigma-Aldrich). Silicone samples
were then submerged in the EDC/NHS solution on a 24-well culture
plate for 1 hour at room temperature, on a shaker at 300 rpm. After
1 hour, samples were removed from the solution and placed in a new
24-well culture well plate. The sample was then submerged in a
concentration of 5ng/mL solution of ligand JAG1 (1277-jg,
rndsystem) for 1 hour at room temperature. Samples were then
thoroughly washed using DH2O for three times. The samples were then
placed in CellCrown (Sigma-Aldrich) inserts and placed in a new
24-well culture plate. In parallel, this procedure was done on Nunc
polystyrene well plate and non-modified silicone. Cell culturing
& Extraction:
[0164] WPMY-1 cells (ATCC) were detached using 0.25% Trypsin/EDTA
(Thermofisher) and a cell suspension of 2.5.times.10.sup.5 cells
was made using DMEM medium including 10% FBS, 1% Glutamax and 1%
penicillin/Streptomycin. A volume of 400 .mu.l cell suspension
(1.0.times.105 cells) was then placed on all samples (including the
controls). Each well was then filled up to 1 mL DMEM medium. The
well plate was then placed in an incubator (37.degree. C/5% CO2)
for 48 hours. RNA was extracted using a RNeasy mini plus kit
(Qiagen).
[0165] Pathway analysis: The Philips Pathway analysis (pathway
analysis as disclosed herein) can be used to determine the activity
of cell signaling pathways in human cancer tissue. The test is
usually based on RT-qPCR expression analysis of multiple target
genes for different pathways (AR, ER, AP1/MAPK, FOXO, TGB.beta.,
HH, Notch). Pathway activity scores will be calculated based on
mRNA expression values using a computational model. The RNA of the
samples is amplified using a one-step RT-qPCR kit. The extracted
RNA sample will be used for RT-qPCR amplification on the
Multi-Pathway-Plate (Philips). Therefore, the PCR sample is mixed
with PCR reagents and aliquotted in the PCR plate containing the
pre-spotted primer sets for the pathway target genes. After running
the PCR program, Cq values (expression values) are used as input
for calculating the Pathway activity scores using the Pathway
computation software (Philips Pathway analysis as disclosed
herein). This methodology was used to infer Notch pathway activity
(also referred to as pathway activity score) in the present
experiment.
[0166] Results: FIG. 4 shows the results from the ligand-induced
Notch pathway activation in (from left to right) polystyrene (PS)
(left), modified silicone (MS)(middle) and regular silicone (S)
(right) culture plates. The NOTCH ligand-coupled (coated)
polystyrene culture plate (left) served as a positive control, the
non-modified silicone culture plate (right) as negative control.
Each ligand-coated/coupled experiment (right bar of each of the
three pairs) is accompanied by a respective experiment without
ligand (left bar of each of the three pairs). Pathway activity is
indicated as pathway activity score, whereby the higher the score
the higher the Notch pathway activity. As can be gathered from FIG.
4, upregulation of the Notch pathway was seen in the positive
control (polystyrene well plate, coated with NOTCH ligand) as in
the modified silicone samples with coupled NOTCH ligand, and not in
the control samples without ligand. No upregulation of the Notch
pathway activity was seen in the negative control (non-modified
silicone), with or without NOTCH ligand coating procedure.
[0167] Other variations to the disclosed embodiments can be
understood and effected by those skilled in the art in practicing
the claimed invention, from a study of the drawings, the
disclosure, and the appended claims.
[0168] In the claims, the word "comprising" does not exclude other
elements or steps, and the indefinite article "a" or "an" does not
exclude a plurality.
[0169] A single unit or device may fulfill the functions of several
items recited in the claims. The mere fact that certain measures
are recited in mutually different dependent claims does not
indicate that a combination of these measures cannot be used to
advantage.
[0170] A computer program may be stored/distributed on a suitable
medium, such as an optical storage medium or a solid-state medium,
supplied together with or as part of other hardware, but may also
be distributed in other forms, such as via the Internet or other
wired or wireless telecommunication systems.
[0171] Any reference signs in the claims should not be construed as
limiting the scope.
* * * * *
References