Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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"DEVICE AND METHOD FOR MULTIDIMENSIONAL CELL CULTURE"
Field of the invention
The present invention lies in the field of molecular cell biology and provides
cell culture devices and
methods for multidimensional cellular analyses, more particularly three-
dimensional (3D) and four-
dimension (4D) based device and methods. Methods of making such devices are
also provided. The cell
culture methods and devices are useful in drug discovery and development,
clinical trials, therapy decisions
and a focused Patient Genome/Cancer Genome Treatment-Outcome data.
Background
Human physiology, pathology and tissue based analyses have until recently been
done on two-dimensional
(2D) cell culture methodology, which has played a pivotal role in furthering
various developments in the
research areas of developmental biology, tissue morphogenesis, disease
mechanisms, drug discovery, tissue
engineering, regenerative medicine and organ printing. There have been
significant discoveries made and
utilized based on this methodology and that have benefitted the global
population. However, as the research
capabilities are undergoing a tremendous outlook change and paradigm shift, a
multitude of gaps and
insufficiencies associated with 2D cultures become apparent, especially with
respect to the inability of 2D
cultures to emulate in vivo conditions and provide physiological relevance. In
the field of cancer diagnosis
and medicine particularly, the gaps presented by differences between the in
vivo and in vitro scenarios are
a well-recognized challenge. The 2D cell-based assays have shortcomings such
as design flaws, 3D spatial
issues, difficult accessibility, and generally do not represent the effective
3D in vivo milieu. While rapid
strides have been taken in the last few years to bridge this gap and genomic
tools have been tried, they have
not been fully efficient to address the complexities that exist in the patient
or animal model. The gap
between data generated in 2D cell based/functional assays are often 2-20-fold
in dosage strengths of drugs
dose titration studies that are relied upon as a rapid cut off for candidate
molecules. Thus, they do not
represent a real-world determination of the potential and efficacy of
molecules, even further, as the animal
models have their own flaws and dynamics.
The scientists in last few years have endeavoured to create in vitro or
artificially, an environment for the
cells in which these cells are able to grow and interact with their
surroundings in three dimensions (3D).
Three-dimensional (3D) cell culture is now attaining the status of new norm in
the cell culture space in
biomedical research field. According to current practices, 3D cultures are
grown in cell culture bioreactors
or miniaturized plate-based systems/ capsules in which the cells can grow into
spheroids, or 3D cell colonies
[Goodman et al, Mierose. Mieroanal. 22 (Suppl 3), 2016]. Cell culturing of
mammalian and human cells
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in 3D to create tissue like organs is revolutionizing analysis in cell culture
techniques and has found
application in different fields with a promising future growth.
The key to successfully growing a homotypic or a heterotypic 3D tissue culture
model is to mimic the
physiologic, histologic, and functional properties of the respective tissues.
The homotypic system includes
pure cell lines and the heterotypic system includes, for example, biopsy
samples of real tumor which contain
cells of mixed lineages. The 3D cell culturing methods and further development
of various applications
have made significant inroads into various medical, pharmaceutical and
biotechnology-based applications.
Numerous studies are ongoing in the field of cancer, stem cell research, drug
discovery, and regenerative
medicine, to name a few [Report ID: GVR-1-68038-091-0, Published Date: Jun,
2018]. Hospitals,
pharmaceutical companies, research institutes and laboratories are adopting 3D
cell culture methodology
and its derivations to obtain better outputs and the adoption rate is posed to
increase rapidly in the next
decade. The establishment of 3D cell culturing methods has been based on use
of either the scaffold-based
platforms, scaffold-free platforms, gels, bioreactors and/or microchips.
Various scaffold-based platforms
have been described in literature which are macro-porous, micro-porous, nano-
porous, or solid scaffolds.
However, these systems are not entirely efficient in terms of being tedious to
produce and use, and are
excessively time consuming, unstable over long periods, low throughput, may
have biocompatibility issues
with tissue samples, may pose sample retrieval challenges, etc. (Archana Swami
et al., 3D Tumor Models:
History, Advances and Future Perspectives; Future Oncology, May 2014).
The present invention addresses the existing challenges in the prior art to
achieve a satisfying functional
outcome so as to provide a multidimensional system and method that closely
simulates the inner micro-
and macro-scale features of the engineered tissue/s.
Summary
The present invention provides a high throughput device and method for
multidimensional cell culture,
more particularly three-dimensional (3D) and four-dimensional (4D) devices and
methods. The device and
method of the present invention comprise growing cells as spheroids and/ or
tissueoids on a non-woven
fabric base matrix system to create 3D tissue like structures. The present
invention &so provides methods
and devices for analysis of drug sensitivity of cells. Further, the invention
provides a device for
characterizing and analysing features of growth and drug sensitivity of cells,
characterization of a variety
of cell strains and biopsy samples.
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The devices and methods of the present invention address the challenges faced
in applying the existing
2D/3D systems and present a wide range of industrial applications particularly
in cancer drug development,
clinical trials, regenerative medicine, and personalized medicine assays,
among others.
An aspect of the invention provides a device for growth of cells comprising a
plurality of sterile culture
chambers, each chamber containing a sterile non-woven fabric base matrix
system for receiving and
supporting an inoculum selected from the group of: a spheroid from a hanging
drop culture, a volume of a
cell culture, and a primary culture of a biopsy or an explant, each chamber
having a bottom and sides for
holding culture medium, the base matrix system and cells, for growth of the
cells in three dimensions (3D).
In general embodiments of the device, the fabric of the base matrix system
includes a non-woven matrix of
polymer or copolymer fibers consisting of at least one selected from the group
of: polyethylene
terephthalate (PET), polypropylene (PP), polystyrene (PS), polyamide (PA),
polyethylene (PE), PBT
(Polybutylene terephthalate), glass fiber, acrylic resin, and cotton.
Typically, the fabric of the base matrix
system has a density of approximately 10-50 gm/m2 and a thickness of at least
about 0.05 mm and less than
about 5 mm.
In general embodiments, the cells are mammalian in origin, primarily human
cells, but growth of cells in
the device is visualized to be possible for cells of other eukaryotic
organisms including avian, reptilian, and
eukaryotic micro-organisms such as yeasts. In additional embodiments, the
device is employed using cells
that in origin are selected from a plant, a fungal species, and a bacterial
species.
In another aspect of this invention, the device helps generate a tissueoid
from cells of different origin in
comparatively less time for performing further screening studies, wherein the
tissueoids can be visually
seen in less than 72 hours or less than 48 hours or even less than 24 hours.
In another aspect of this invention, the device helps create an extracellular
and intracellular architecture of
the tissueoid that contains at least one component of an extracellular matrix,
such that the extracellular
matrix includes production and further proliferation of collagen or vascular
tubules and intracellular matrix
includes at least one intracellular microscopically visible structure such as
tubulin and/ or actin.
Another aspect of the invention herein provides a method of making a device
for three dimensional growth
of cells, the method including steps of: providing samples of cells selected
in origin from a biopsy of a
patient, an explant from a biopsy, a cell culture in a tissue culture plate,
and hanging drop cultured cell
spheroids, to obtain a resulting multicellular inoculum or a plurality of
multicellular inocula; transferring
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the inoculum or inocula to a corresponding plurality of culture vessels each
containing a non-woven fabric
base matrix system and growth media; and, incubating the vessels to obtain the
three dimensional spheroids
of cells in the device. In a particularly advantageous embodiment of the
method, each sample of cells
contains less than about 1,000 cells, less than about 500 cells, or even less
than about 250 cells, or even less
than about 25 cells. Thus a single sample such as a biopsy sample or a hanging
drop culture provides a
plurality of aliquots of inocula for a plurality of culture vessels. The cells
in the device have been
demonstrated to remain viable and retain functionality for at least about 30
days, or at least about 60 days,
or at least about 90 days or at least about 250 days or at least 380 days and
even substantially longer.
Another aspect of the invention herein provides a method of use for analysis
of cell drug response or
sensitivity of a device for three dimensional growth of cell spheroids on a
non-woven fabric support base
matrix system, the method including steps of: contacting at least one test
chamber of spheroids with at least
one concentration of a drug and comparing growth and viability of the cells in
the spheroid with growth in
a control chamber absent the drug but otherwise identical, such that the
spheroids are cultured from a
patient or from a disease cell line or from a disease model animal. In a
particular embodiment, at least one
concentration is a plurality of concentrations of the drug in a corresponding
plurality of test chambers;
and/or, the drug is a plurality of drugs in a plurality of test chambers.
Generally, the test chamber and the
control chamber contain spheroids/tissueoids cultured from biopsy tissue from
the patient of a tumor or a
cultured cell line. An additional control chamber contains a
spheroid/tissueoid that contains non-tumor
normal cells from the patient. In a specific embodiment for a patient having a
tumor, the drug is an anti-
cancer chemical agent or an anti-cancer antibody or binding protein. For the
patient having the tumor, an
embodiment of the method includes at least one test chamber that contains a
combination of two or more
drugs. In alternative or additional' embodiments, at least one test chamber
contains a drug selected from:
anti-bacterial, anti-inflammatory, anti-viral, anti-helminthic, and anti-
psychotic. An embodiment of the
method comprises continuing growing the spheroid/ tissueoid and analysing cell
functions and responses
for at least about 30 days, or at least about 60 days, or at least about 90
days or at least about 250 days or
at least about 380 days or even over a year.
Accordingly, an aspect of the invention provides a device for growth and drug
sensitivity characterization
of cells from a subject with cancer, the device comprising a plurality of
sterile culture chambers, each
chamber containing a sterile non-woven polyethylene terephthalate (PET) fabric
base matrix system for
receiving and supporting an inoculum of subject cells selected from the group
of: a spheroid from a hanging
drop culture, a volume of a cell culture, and a primary culture of a biopsy,
and a test plurality of cultures
originates from cancerous tissue from the subject, and a control culture or
biopsy originates from normal
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tissue from the subject, each chamber having a bottom and sides for holding
culture medium, the base
matrix system, and cells, for characterization of growth and viability of the
cells in three dimensions (3D)
under a set of variable medium constituents. In a particular embodiment the
device further includes the
cultured cells in the chambers. For example, the sterile culture chambers are
wells in a multi-well culture
dish, for example, a 24 well culture dish or a 96 well culture plate.
An aspect of the invention provided herein is a set of one or more tissueoid
cell cultures produced by the
methods herein.
Another aspect of the invention herein provides a cell culture and artificial
tissue production device
comprising at least one or a plurality of sterile culture chambers, each
chamber containing cells and a sterile
non-woven polyethylene terephthalate (PET) fabric base matrix system for
receiving and supporting an
inoculum of cells, in which the cells are selected from the group of:
spheroids from a hanging drop culture,
volumes of a cell culture, and primary cultures of a biopsy, each chamber
having a bottom and sides for
holding culture media, the base matrix systems, and the cells, and each
chamber having a port for addition
of fresh culture medium and a drain for depletion of spent medium. In general,
the cell origin is avian or
mammalian. For example, the cell origin is muscle, epithelial or other tissue.
Brief description of the drawings
Fig. IA-Fig. 1C are a set of photographs of microscopic images of three-
dimensional organization of non-
woven fabric as a base matrix system either empty (Fig. 1A) or in presence of
MCF-7 (breast cancer cell
line) tissueoids (Fig. 1B and Fig. 1C) demonstrating system provided herein to
form tissue like structures.
The base matrix system is a fabric mat which is made with spun-bond
technology. The images (Fig. 1B &
Fig. 1C) illustrate growth of tissueoids on the top of the AXTEX-4D base
matrix system in a 3D fashion.
In order to make a 3D culture, spheroids were formed using hanging drop method
followed by growth on
base matrix system resulting in tissueoid formation, using the methods and
systems provided herein.
Fig. 1A shows the photograph of AXTEX-4D base matrix system in its original
form without any cells or
tissueoids grown on AXTEX-4D, observed by scanning electron microscope (SEM).
(Magnification 100X)
Fig. 1B shows tissueoid derived from breast cancer cell line MCF-7 grown on
AXTEX-4D base matrix
system, observed by compound microscopy. (Magnification 40X)
Fig. IC shows image of breast cancer tissueoid derived from MCF-7 cell line
grown on AXTEX-4D base
matrix system, observed by SEM. (Magnification 350X)
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Fig. 2 is a set of photographs observed by phase contrast microscopy, in which
HT-29 cell line grown on
the base matrix system to form the 3D tissueoids. Growth of tissueoids is
observed on base matrix system
with different densities (19 g/m2, 20 g/m2, 30 g/m2, 35 g/m2) of fabric mat
Fig. 3A-Fig. 3B are set of photographs of spheroids and or tissueoids showing
interactions of cells with
each other, resulting in cell growth in three dimension on AXTEX-4D system.
Fig. 3A shows images of spheroids (upper panel) derived from MCF-7 and HUVEC
cell lines or spheroids
cultured on AXTEX-4D base matrix to obtain tissueoids (lower panel).
Photographs are observed by phase
contrast microscopy. (Magnification 10X).
Fig. 3B is a set of images, showing structural and spatio-temporal
organization of tissueoid growth on the
AXTEX-4D system. Cell-cell connectivity and organization with extracellular
matrix were observed,
including 3D tissue like organization, cell-cell connection and interaction of
organization of biopsy
specimen taken from colon cancer, directly grown on the base matrix system and
grown as tissueoid on
AXTEX-4D system. Photographs were taken using SEM. (Magnification 1500X,
7000X).
Fig.4 shows tissueoids of transformed cell lines HEK-293 and CHO-Kl observed
by scanning electron
microscopy.
Upper panel: shows the three-dimensional organization of human embryonic
kidney cell line (HEK-293)
tissueoids grown on AXTEX-4D base matrix system (Magnification 1000X, 1500X)
Lower panel: shows the three-dimensional organization of Chinese hamster ovary
cell line (CHO-K1)
tissueoids grown on AXTEX-4D base matrix system (Magnification 1000X, 1500X).
Fig. 5 shows scanning electron microscopy images of biopsy explant taken from
lung cancer, and grown
as such on the base matrix system. The cells from biopsy were cultured on
AXTEX-4D base matrix and
growth resulted in tissueoid production without any prior treatment given to
the cells. (Magnification
1000X, 1500X)
Fig. 6 shows confocal microscopy images of tissueoid on AXTEX-4D system taking
PC3 cell line as an
example, stained with calcein AM showing proliferation/growth as well as
viability of tissueoids observed
on different days i.e. 3, 25, 108 and 250 days. To determine cell viability,
calcein AM cell-permeant dye
was used.
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Fig. 7A-Fig. 7F show the formation of extracellular matrix in 2D monolayer and
3D tissueoids using MCF-
7 cell line as an example on AXTEX-4D system. Staining was performed with anti-
collagen type-I antibody
(green) in 1:50 dilution, DAPI (blue- nuclei stain) on Day 7 and observed by
fluorescence microscopy. The
ECM formation in the tissueoids formed on AXTEX-4D system was observed to be
more contiguous as
seen in image (Fig. 7A-Fig. 7F) compared to that of cells culturedin 2D
monolayer. (Magnification 10X).
In 2D culture, the figures represent Fig. 7A- nuclei staining with Hoechst,
Fig. 7B- collagen staining with
anti-collagen antibody and Fig. 7C- is the merged image. In tissueoids
analysis, the figures represent Fig.
7D- nuclei staining, Fig. 7E-collagen staining, Fig. 7F-merged image.
Fig. 7G-Fig. 7H are the images of entire mass of tissueoids generated from MCF-
7 cell line grown on
AXTEX-4D system. Staining was performed with anti-collagen type-I antibody
(green) in 1:50 dilution,
DAPI (blue- nuclei stain) on Day 7 and observed by confocal microscopy.
(Magnification 10X)
Fig. 8 compares the formation of intracellular matrix in 2D monolayer and 3D
tissueoids using MCF-7 cell
line as an example on AXTEX-4D system. Staining was performed with anti-
phalloidin antibody (Red) in
1:1000 dilution, DAPI (blue- nuclei stain) showing that tissueoids grown on
base matrix system contained
cytoskeletal components. The cytoskeletal organization in the tissueoids was
observed to be more
contiguous as seen in image above (Fig. 8) compared to that of cells
cultured_in 2D monolayer.
(Magnification 10X)
Fig. 9A-Fig. 9E show tissueoids generated from various cell numbers of MCF-7
cells, with a range between
¨250 to ¨25 cells using phase contrast microscope. Tissueoids were grown on
AXTEX-4D system. Fig.
9A, 9B, 9C, 9D, 9E are images from phase contrast microscopy study (10X
magnification) and Fig. 9F is
an image using scanning electron microscope for ¨ 25 cells grown on AXTEX-4D
system. (Magnification
1500X). Inocula in a range of cell numbers from 5000 cells and less were
evaluated and the data shown
here depict successful growth on the AXTEX-4D system of about 250 cells to as
less as 25 cells.
Fig. 10A-Fig. 10B is a set of photographs showing morphological
characteristics of MCF-7 cells either
grown as 2D monolayer culture or as tissueoids grown on AXTEX-4D system.
Fig. 10A represents morphology of MCF-7 cells treated with or without
doxorubicin after 3 days grown in
2D culture. Dose-dependent growth inhibition was observed in treated group
compared to that of cells
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cultured in presence of DMSO (vehicle control). Vacuoles were observed even at
1 tM doxorubicin in 2D
culture indicating the sensitivity of cells towards doxorubicin treatment
which eventually led to cell death.
The insets in the upper panel images are shown as a magnified view in the
lower panel images.
Fig. 10B is a set of photographs that show sensitivity of MCF-7 tissueoids
against doxorubicin after 3 days
of incubation. Growth of tissueoids generated from MCF-7 cell line was not
inhibited at 2.5 uM
concentration of doxorubicin and was comparable to the growth observed in the
tissueoid growing without
the drug in presence of vehicle control (DMS0). Partial growth inhibition of
tissueoid was observed at 5 M
concentration. At higher concentration (5 uM), tissueoids did not disintegrate
from the AXTEX-4D base
matrix system but remained attached to it, though shrinking of the tissueoid
was visible.
Fig. 11A-Fig. 11B are set of bar graphs that describe the sensitivity of MCF-7
cells either grown in a 96
well plate as a monolayer or tissueoid on AXTEX-4D system towards doxorubicin
at indicated
concentrations in presence or absence of bevacizumab antibody.
Fig. 11A shows drug sensitivity analysis using three different concentrations
of doxorubicin in both 2D
monolayer culture and 3D tissueoid system for 48 hrs. The viability of the
cells in each of monolayer culture
(2D) and in tissueoid (3D) was evaluated by analysing viability using
prestoblue. The data is expressed in
relative fluorescence unit (RFU) and normalized with the vehicle control as
100% viability. Resistance of
the drug activity was observed in tissueoids grown on AXTEX-4D system even at
1 aM of doxorubicin
concentration (-80% viability). At the same concentration (1 uM of
doxorubicin), cells cultured in 2D
monolayer showed 35% viability.
Fig. 11B shows the combinatorial effect of doxorubicin and bevacizumab on VEGF-
165 induced MCF-7
cell proliferation grown as tissueoids. MCF-7 tissueoids were cultured in
wells of a 96 well plate. Cells
were serum starved for about 5 hrs and subsequently treated with 10Ong/m1 of
VEGF-165 either alone or
in combination with laM doxorubicin and 25 g/m1 of bevacizumab for 6 days.
Viability of the cells was
analysed using prestoblue. Tissueoids grown on AXTEX-4D system showed greater
efficacy of
combination effect of both drugs (about 57%) as compared to monotherapy (as
shown in Fig. 11A) to
prevent cell growth.
Fig. 12 represents phase contrast image of tissueoids of HT-29 before (Fig.
12A) and after treatment (Fig.
12B) with cytokine TNF-a (20 ng/m1) in combination with IFN-y (0.5 ng/m1) for
16-18 hrs. Intact tissueoids
became fragmented and dislodged from AXTEX-4D base matrix system after
treatment with cytokine
demonstrating impact of cytotoxicity.
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Fig.13 is a set of photographs that shows the duration in length of time (in
days as indicated in each panel)
during which the tissueoids remained in culture. Using phase contrast
microscopy, the longevity of
tissueoids derived from HepG2 and PC3 cells was observed. Different fields on
different days were
captured and increased number of cells with increasing density was observed.
PC3 tissueoids are viable in
culture as of the date of filing of the present application. (Day 364).
Fig.13A -Fig. 13B Viability of Hep-G2 tissueoids was observed until day 82.
Fig.13C-Fig. 13D Viability of PC3 tissueoids was observed until day 364.
Fig. 14 is a set of photographs that shows mono, co and tri culture of three
cell lines by adding cell
suspension of transformed fibroblast cell line (NIH-3T3), endothelial cells
(HUVEC) and breast cancer cell
line (MCF-7) and grown as 2D monolayer culture or as tissueoids on base matrix
system AXTEX-4D. Co-
culture of each of the combinations were analysed by taking either breast
cancer cell line (MCF-7) and
endothelial cells (HUVEC) or endothelial cells (HUVEC) and fibroblast (NIH-
3T3) in 1:1 ratio
respectively. For tri-culture NIH-3T3, HUVEC and MCF-7 cells lines were -added
in 2:1:1 ratio. The
spheroids were formed for all combinations and cultured in 2D monolayer as
well as on the AXTEX-4D
base matrix system. Attachment of spheroid was observed within 24 hrs on the
AXTEX-4D base matrix
system and all combinations were observed to have grown further, as
tissueoids.
Fig. 14 (upper panel) shows monolayer culture of spheroids made of HUVEC,
HUVEC: MCF-7, HUVEC:
3T3 and HUVEC: MCF-7:3T3 grown on 2D format.
Fig. 14 (lower panel) shows the tissueoids of HUVEC, HUVEC: MCF-7, HUVEC:3T3
and HUVEC: MCF-
7:3T3 grown on AXTEX-4D base matrix system.
Fig. 15 is a set of photographs that show tissueoids of HEK-293, NIH-3T3 and
PC3 grown on AXTEX-4D
system. Fig. 15 shows minimum time taken for tissueoids to have adhered and
have initiated growth on the
AXTEX-4D system. Tissueoids of HEK-293 and NIH-3T3 cell lines took less than24
hrs for attachment
and proliferation of cells on AXTEX-4D system whereas tissueoids of PC3 took
approximately 48 hrs to
adhere and further grow on AXTEX-4D system.
Fig. 16A-Fig. 16B is a set of photographs that shows application of AXTEX-4D
system as cell factory.
Adherent CHO-DG44 stable cell line expressing tocilizumab is growing as a
tissueoid on AXTEX-4D base
matrix system and secretion of monoclonal antibody Tocilizumab is observed in
the culture supernatant, as
analysed by SDS PAGE.
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Fig. 16A shows growth of tocilizumab expressing CHO-DG44 cells as tissueoids
on AXTEX-4D.
Fig. 16B shows expression analysis of tocilizumab by non-reducing SDS-PAGE.
Briefly, culture
supernatant was taken out from different day's culture from cells growing on
petri-plate as 2D culture and
tissueoids grown on AXTEX-4D system. Tissueoid grown on AXTEX-4D base matrix
system, allowed
increased number of cells in a more compact space, with increased longevity (6
days in monolayer and 26
days as tissueoids when the samples were taken out for analysis) and better
productivity.
SDS PAGE analysis showing expression of monoclonal antibody (Tocilizumab) in
adherent CHODG44
cell line in 2D as well as 3D format; each lane of 10% SDS-PAGE was loaded
with a different sample;
Lane 1: Prestained protein marker, Lane 2: Positive control (1 g), Lane 3: Day
6 sample of 2D culture
supernatant, Lane 4: Day 6 sample of tissueoids supernatant, Lane 5: Day 12
sample of tissueoids
supernatant, Lane 6: Day 18 sample of tissueoids supernatant, Lane 7: Day 26
sample of tissueoids
supernatant'. Equal number of cells were seeded on 2D as well as 3D format.
After 6 days 2D culture was
terminated due to confluency of culture of tissueoids was sustained till day
26. SDS-PAGE revealed that
tocilizumab antibody expression was observed on day 6 in 2D monolayer culture,
whereas no expression
was observed in tissueoids grown on AXTEX-4D on day 6. However, the expression
of tocilizumab in 3D
culture was observed to have increased as a function of time of incubation in
days from day 12 to day 26.
Fig. 17A- Fig.17B are set of photographs that show growth of endothelial cells
HUVEC as tissueoids on
AXTEX-4D base matrix system in presence (Fig. 17B) and absence of VEGF-165
treatment (Fig. 17A) for
72 hrs and observed by phase contrast microscopy. Fig. 17A shows attachment of
spheroid to the base
matrix system with minimal proliferation. Fig. 17B shows proliferation of
cells along with tube like
structure formation demonstrating that angiogenesis was observed in AXTEX-4D
system. Fig 17. C
(magnified view of tissueoid with VEGF treatment) is the magnified view
depicting closer look at the tube-
like structure.
Fig. 18 is a drawing of an embodiment of the invention which is a device with
the 3D and 4D elements.
Fig. 19 is a drawing that shows various uses of the devices provided herein
and relative advantages.
Detailed description of the embodiments
The following description with reference to the accompanying drawings is
provided to assist in a
comprehensive understanding of exemplary embodiments of the invention. It
includes various specific
details to assist in that understanding but these are to be regarded as merely
exemplary.
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While the invention is susceptible to various modifications and alternative
forms, specific embodiments
thereof have been described in detail below. It should be understood, however
that it is not intended to limit
the invention to the particular forms disclosed, but on the contrary, the
invention is to cover all
modifications, equivalents, and alternatives falling within the spirit and the
scope of the invention. In
addition, descriptions of well-known functions and constructions are omitted
for clarity and conciseness.
The terms and words used in the following description and claims are not
limited to the bibliographical
meanings, but, are merely used by the inventors to enable a clear and
consistent understanding of the
invention. Accordingly, it should be apparent to those skilled in the art that
the following description and
embodiments of the present invention are provided for illustration purpose
only and not for the purpose of
limiting the invention as defined by the appended claims and their
equivalents.
Features that are described and/or illustrated with respect to one embodiment
may be used in the same way
or in a similar way in one or more other embodiments and/or in combination
with or instead of the features
of the other embodiments.
It should be emphasized that the term "comprises/comprising" when used in this
specification is taken to
specify the presence of stated features, integers, steps or components but
does not preclude the presence or
addition of one or more other features, integers, steps, components or groups
thereof.
Accordingly, the present invention relates to cell culture systems for
multidimensional, particularly, 3D/4D
systems for cellular and molecular studies. A device encompassing the novel
3D/413 tissue culture models
is also provided. Further, the present invention provides methods of preparing
the said multidimensional
cell culture systems.
An objective of the present study is to provide cell culture systems for
multidimensional tissue model
analyses. More particularly, 3D/4D tissue culture and tissueoid generation
system for cellular and molecular
analyse and further applications of it.
Yet another objective of the present invention is to provide method of
preparing the aforesaid systems and
culture devices.
Yet another objective of the present invention is to provide a high throughput
device for growth of cells in
which it is containing a plurality of sterile culture chambers, each chamber
containing a sterile non-woven
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fabric base matrix for receiving and supporting an inoculum selected from the
group of a spheroid from a
hanging drop culture, or direct suspension of cells derived from tissue, a
volume of a cell culture derived
from cell lines, and a primary culture of a biopsy or an explant, each chamber
having a bottom and sides
for holding culture medium, the base matrix and cells, for growth of the cells
in three dimensions (3D). The
devices provided herein contain a non-woven mat of polymer fibers consisting
of at least one selected from
the group of: PET, PP, PBT, glass fiber, and cotton.
In another embodiment, devices are provided, the fabric of the base matrix has
a density of in range of
approximately 10 gm/m2 and 50 gm/m2, for example 19-25 gm/m2 and a thickness
of at least about 0.05
mm and less than about 5 mm, for example 0.12 mm. The thickness of the fibers
is 0.5-10 dtex, for example
2.5-3.0 dtex and the porosity in range of 20-80 micron.
In another embodiment, the device of the present invention is used to grow
cells selected from a mammalian
species. In embodiments, the mammalian cells are human, such as established
cell lines or fresh biopsy
samples from a patient, or are other mammalian cells such as Chinese Hamster
ovary derived cells (CHO
and CHO derived cells).
The tissueoids that have been grown successfully using this technique include
the following cancerous cell
lines: MCF-7: breast cancer cell line from an adenocarcinoma; HepG2: liver
carcinoma of epithelial cells;
PC3: prostate cancer cell line from an adenocarcinoma; and A375: skin melanoma
which is an epithelial
cell line.
The following non-malignant cell lines have been successfully grown: as shown
in examples herein CHO
cells (Chinese hamster ovary); HEK-293 (human embryonic kidney cells); and NIH-
3T3 (Fibroblasts).
Primary tissues that have also been successfully used as a source of cells
include: breast cancer tissue from
a tumor; colon cancer from a tumor; gastric cancer from a tumor; lung cancer
from a tumor and; thyroid
cancer from a tumor.
In general embodiments of the device, tissueoids grown by the methods herein
produce extracellular
architecture i.e, collagen. Tissueoids growing in this base matrix AXTEX-4D
system were observed to
produce 3D like rearrangement of cytoskeletal elements by analysing expression
of F-actin. Tissueoids
continued to proliferate for an extensive period of time indicating favourable
growth conditions provides
by the device here in.
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An embodiment of the invention provides a method to grow a cell sample
employing as an inoculum a
sample containing less than about 1,000 cells, less than about 500 cells, less
than about 250 cells and even
less than 25 cells. The cells are derived from stable cell lines or from
living tissues such as tumor biopsies
that are cultured ex vivo using the device and methods herein.
In another aspect, the invention provides a method of use for analysis of cell-
drug sensitivity of a device
for three dimensional growth of tissueoids cultured from cells of a patient,
on a non-woven fabric support
base matrix in which it is contacting at least one test chamber of tissueoids
with at least one concentration
of a drug, and comparing growth and viability of the cells in the tissueoids
with a control chamber absent
the drug but otherwise identical.
In an embodiment, at least one concentration is a plurality of concentrations
of the drug in a corresponding
plurality of test chambers; and/or, the drug is a plurality of drugs in a
plurality of test chambers. The drug
is selected from an anti-cancer chemical agent or an anti-cancer antibody or
binding protein.
In another preferred embodiment, the test chamber and the control chamber
contain tissueoids cultured
from biopsy tissue from the patient of a tumor.
In yet another embodiment, an additional control chamber contains tissueoids
containing non-tumor
physiologically normal cells from the patient is provided.
In another embodiment, at least one test chamber contains a drug or a
combination of two or more drugs.
In a further embodiment, at least one test chamber contains a drug selected
from: anti-bacterial, anti-
inflammatory, anti-viral, anti-helminthic, and anti-psychotic.
In another aspect of the invention, a device for growth and drug sensitivity
characterization of cells from
inocula with cancer is provided. The device comprises a plurality of sterile
culture chambers, each chamber
containing a sterile non-woven polyethylene terephthalate (PET) fabric base
matrix for receiving and
supporting an inoculum of cells. The inocula are selected from the group of: a
spheroid from a hanging
drop culture, a volume of a cell culture, and a primary culture or explant
from a biopsy, such that a test
plurality of cultures originates from cancerous tissue from the inocula, and a
control culture or biopsy
originates from normal tissue from the inocula. Each chamber has a bottom and
sides for holding culture
medium, the base matrix, and cells, for characterization of growth and
viability of the cells in three
dimensions (3D) under a set of variable medium constituents. In a further
embodiment, the cultured cells
are present in the chambers in a multi-well culture dish, for example, a 24
well culture dish or a 96 well
culture plate.
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The present invention also provides a cell culture and ex vivo tissue
production device including at least
one or a plurality of sterile culture chambers, each chamber containing cells
and a sterile non-woven
polyethylene terephthalate (PET) fabric base matrix for receiving and
supporting an inoculum of cells. The
cells may be selected from the group of: spheroids from a hanging drop
culture, volumes of a cell culture,
and primary cultures of a biopsy, each chamber having a bottom and sides for
holding culture media, the
base matrix, and cells, each chamber having a port for addition of fresh
culture medium and a drain for
depletion of spent medium.
The invention provided a device having tissueoids in which the cell origin is
avian or mammalian.
In an embodiment, the cell origin may be selected from muscle, epithelial or
other tissue.
The present invention also provides a use of the resulting production by the
device for a therapeutic artificial
skin or muscle.
In another aspect, the cells of the tissueoids produced by the method and
devices provided herein of the
present invention have longer lifespans, viz., longer period of time of cell
viability compared to what is
reported in the prior art. A viability of up to 250 days has been observed in
the present examples with
different cell lines, as shown in Fig. 6 and further continued growth of cells
has been shown up to 364 days,
as shown in Fig. 13. It has been observed that the tissueoids remain viable
with adequate form and function
for a period of time such as more than 12 months. Various examples have been
conducted with different
cell lines and primary cells and the longevity of the tissueoids has been
reproducibly seen to be substantially
greater than that reported earlier.
In another aspect, the present method and device provides 3D culture assays
that are initiated in less time
(less than 72 hours) than reported previously. Zanoni M et al. forms spheroids
using hanging drop method
by using 2 x 103, 4 x 103, 6 x 103 cells/well, however these spheroids were
reported to need a period of 7
days.
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Time required No. Cell Equivalent Amount of
Amount large
[day] Required [x10 diameter [p.m] spherical
spheroids (500
(range, spheroids p.m)
mean SD, CV, (S I>0. 90)
n)
Magnetic 7 0.5 200-500, Low Low
Levitation* 347 87,
25.1,28
Hanging drop- 7 0.6 200-500, Low Low
359 95,
26.5,38
Pellet 1 20 800-900, High High
Cultures 880 21, 2.4,20
Rotating Waif' 15 40 500-1100, Low' High
Vessel (NASA 897 98,
B ioreactor) 11.0,192
Table 1. Scaffold free techniques for obtaining tumor spheroid models.
rHaisler, W. L. et al. Three-dimensional cell culturing by magnetic
levitation. Nat. Protoc. 8, 1940-1949
(2013). Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M. & Nielsen,
L. K. Method for
generation of homogeneous multicellular tumor spheroids applicable to a wide
variety of cell types.
Biotechnol. Bioeng. 83, 173-180 (2003).7' Johnstone, B., Hering, T. M.,
Caplan, A. I., Goldberg, V. M. &
Yoo, J. U. In vitro chondrogenesis of bone marrow-derived mesenchymal
progenitor cells. Exp. Cell Res.
238, 265-272 (1998).1'Ingram, M. et al. Three-dimensional growth patterns of
various human tumor cell
lines in simulated microgravity of a NASA bioreactor. In Vitro Cell. Dev.
Biol. Anim. 33, 459-466 (1997).]
In the device and the method of the present invention, the spheroids were
generated in about 24 hours or
less. Further, such spheroids were able to bind onto the base matrix in less
than about 24 hours. In another
preferred embodiment of this invention, tissueoids show growth on the base
matrix in less than 24 hours.
Yet another advantage was that the device and method are capable of using
different cell lines. Even the
types of cells which were usually less compact in nature, such as the PC3 cell
line (Prostate cancer) and HT
29 cell line (Colorectal cancer) were observed to display good binding to the
base matrix. The 3D/4D device
and method of the present invention were observed to grow tissueoids that in
function and structure were
observed to be similar to the original tissue across all cell lines tested
herein.
In yet another aspect, the device and method of the present invention yield
results that are comparable to
genomic and proteomic profile of the primary tumor tissue and gives results
that supersede studies done on
monolayer cultures, results of which may be inconsistent and non reproducible.
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In aspects of the present invention, the multi-dimensional physical and
analytical readout properties and
applications of this device/base matrix AXTEX-4D system are the following.
Tissueoids were grown either
as single pure culture, or as combinations of multiple types of cells; as
adherent or in suspension and
combinations thereof, for either support, sustenance or similar to what was
observed in an in vivo network
to simulate the micro-environment in-vivo of the particular organs, tumor, or
interplay of the immune
system against cancers or infections. Multiple combination therapy/ies using
one more combinations of
both chemical and biological drugs was designed, tested and evaluated. Genetic
changes due to impact of
administration of the drugs/combinations in varied doses/dosage forms is
determined as a function of time,
by accelerated studies. Physico-stimulation of the tissueoid to attach,
proliferate and promote accelerated
growth was tested to deliver rapid test results for drugs impact, efficacy
studies, genetic mutations, etc. For
example, the 3D/4D models provided in the present invention is used also to
study the concentration of
various drugs that can be used as effective doses for treatment regimen.
A fast turnaround time for obtaining results allows this model to yield more
effective analysis and benefit
the patients in their clinical outcome.
In another embodiment, the systems of the present invention are used to
establish the drug combination
therapy using both chemical and biological dugs. The systems are envisioned as
useful to select patients
for clinical trials in oncology related trials, to decide on multiple therapy
regimens and multiple
concentrations is studied at the same time i.e., simultaneously, to generate
data from patients having a
disease, and to correlate with data from the tissue growing in vitro, and the
data received after impact of
treatment. Thus, a focused Patient Genome/Cancer Genome Treatment-Outcome
database is generated to
enable effective future treatment regimens, by using the ex vivo AXTEX-4D base
matrix system provided
herein to screen for patients, to identify appropriate efficacious therapeutic
agents for each patient.
In yet another embodiment, the invention provided methods and devices for
validation of tissue like
structures having the ability to support various tumor cell lines in 3D; and a
fourth dimension (4D) as a
function of an extended period of time, and applications thereof in drug
discovery and other clinical
analyses, diagnosis etc.
The device encompassing the 3D tissue system and its configuration is
presented in Fig. 18 which
demonstrates a 3D tumor base matrix system central to the cubic design. In an
embodiment, the device of
the present invention is provided as a 4D base matrix system which is used for
studies on tissues under in
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situ conditions which allow efficient monitoring and evaluation of input and
device output as a function of
time.
The dimension of time when included in execution of cell culture in vitro,
provides methods of quicker
diagnosis of disease as compared to existing methodologies. Further, it also
increases the window of trial
for different analyses due to increased longevity of the tissueoids created by
the methodology described
herewith. The AXTEX-4D base matrix system envisaged in the present invention
to be used for longer
periods of time is pictorially depicted in Fig. 6.
In another important aspect of this invention, the device helps generate a
tissueoid from cells of different
origin in comparatively less time for performing further screening studies,
wherein the tissueoids can be
visually seen in less than 72 hours or less than 48 hours or even less than 24
hours.
An important observation during the studies and thus an important aspect is
that there is evidence of tubule
like structures growing and thus it can possibly be said that the present
invention provides an angiogenesis
model to study antiangiogenic drugs/ assays and other applications
In an embodiment, the systems of the present invention are useful as a source
of material in determining
the proteomic and genomic profile.
In yet another embodiment, the device is used as a biotransformation reactor,
for example to generate high
value proteins such as antibodies during a time course extending for months.
In yet another embodiment is provided a method of determining an efficacious
treatment or regimen of
treating diseases like cancer.
In yet another embodiment the system of the present invention can be used as a
cell factory/ bioreactor, to
grow large cultures and produce therapeutics/ antigens/ vaccine candidates
etc.
Overall, the systems of the present invention are more rapidly growing,
robust, viable and sustainable for a
longer time, with close representation of tissue like structure and function.
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Examples
The present invention is described below in further detail with examples and
comparative examples
thereof, but it is noted that the present invention is by no means intended to
be limited to these examples.
Example 1: Compounds and reagents
Compounds and reagents that were used for drug sensitivity analysis using the
device and methods
provided as a base matrix system AXTEX-4D. Doxorubicin, Cisplatin, Colchicine,
Paclitaxel and DMSO
were purchased from Sigma. These drugs were tested for sensitivity/ resistance
of cells of different cancer
cell lines grown as 2D (Monolayer) and as 3D (On the AXTEX-4D base matrix
system as tissueoids)
formats and the data were compared. Exemplary data are shown in Figs. 10 and
11.
Example 2: Cell lines and tumor analyses
The various human cancer cell lines (such as MCF-7, HepG2, PC3, HT29) were
obtained from the
American Type Culture Collection (ATCC, Rockville MD) A375 and CHO-Kl cell
lines were received
from NCCS, Pune, India. HUVEC was obtained from Lonza. MCF-7 and HepG2 were
cultured in EMEM
(Sigma-Aldrich, St; Louis, MO, USA). PC3 and CHOK-1 was cultured in F12K
(Sigma-Aldrich, St; Louis,
MO, USA) HT-29, A-375, NIH-3T3, HEK-293 cells were cultured in DMEM (Sigma-
Aldrich, St; Louis,
MO, USA). HUVEC cells were cultured in EBM-2 basal medium and EGM-2 Single
Quots supplements.
Fig. 1, 2 and 3A and 4 are representative photographs showing growth of cell
lines on 3D base matrix
system to form tissueoids. All the adherent cell lines were cultured in
presence of 10% FBS (Gibco) and
supplemented with 2 mM glutamine (Sigma- Aldrich, St; Louis, MO, USA). Cells
were cultured at 37 C
humidified condition with 8% CO2 under static condition.
The morphological appearance of tissueoids for each of the cell lines was
analysed as phase
contrast and SEM images in (Fig. 1, Fig. 2. Fig. 3A, Fig. 4).
Generation of tissueoids using primary tumor biopsy: Tumor biopsy samples for
each of colorectal,
gastric, lung and thyroid carcinoma were collected from pathology specimens,
transported for culturing,
and analyzed ex vivo in cell culture lab. Tissue was rinsed with 1X PBS
(without Ca ++ and Mr) thrice and
sliced with a scalpel into smaller pieces and further processed with the
plunger in order to separate and
isolate the cells. Cells were cultured then in DMEM media containing 2 mM
glutamine 2X antibiotic
solution (Penicillin and Streptomycin, Himedia) and 20% FBS. Tumor tissue
specimens were taken as
suspension culture or as explant and grown on AXTEX-4D system. The growth of
tumor tissue on the
AXTEX-4D base matrix system is shown as an example in. Fig. 3B and Fig. 5.
Spheroids and Tissueoids formation. Unless otherwise indicated, spheroids were
formed by using hanging
drop method. This further resulted into formation of tissueoids. Fig. 3A
(upper panel), showing
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development of a spheroid generated by the hanging drop method and Fig. 1,
Fig. 2, Fig. 3A (lower panel),
Fig. 4.is a set of representative photographs of tissueoids growing on the
AXTEX-4D base matrix. The
process for the spheroid formation of various cell lines is described below:
Briefly, cells were seeded at approximately 80% confluency the day before
making hanging drops.
After trypsinization, cells were resuspended in an appropriate volume of
respective media and the process
of hanging drop formation was initiated only when the viability of the cells
was more than 90%. Each cell
suspension was made such that 20 IA of the media contained a cell number in a
range of 103- 104 cells. The
drop was pipetted onto the inner surface of a lid of a sterile culture dish
and PBS was filled in the bottom
of the dish. After 24-48 hrs, the inner lid was inverted and the drops were re-
suspended in a fresh media.
Spheroids were analyzed by phase contrast microscopy. Representative
photograph showing development
of a spheroid using the hanging drop method in Fig. 3A (upper panel) and
tissueoids grown on 3D base
matrix system (Fig. 1B, Fig. 1C, Fig. 2, Fig. 3A (lower panel) and Fig. 4.
Scanning electron microscopy. 3D morphology of the cells attached to the base
matrix AXTEX-
4D system was evaluated by SEM analysis (EVO-18 Research, Zeiss) (Fig. 1C,
Fig. 4). Samples (fixing
agents: 2.5% glutaraldehyde and 2% paraformaldehyde in PBS, pH 7.4) were fixed
on the top of a stub,
vacuum dried for 10 mins with 0.1 mbar pressure followed by addition of argon
gas. Samples were coated
with gold particles using Sputter Coater. Coated samples were then analyzed by
scanning electron
microscopy.
Example 3: Preparation of the 3D cell culture system:
A commercially available spun-bound PET material consisting of extruding round
continuous
filaments (Fig. 1A) which are flat bond, was used in the examples herein. The
fabric used is a non-woven
mat of endless polymer fibers. The density of the fabric is approximately 19-
35 gm/m2 with a porosity of
approximately 65 micron. In order to make a tissueoids, spheroids were
prepared using hanging drop
method and these were cultured for growth on the top of the base matrix (Fig.
1B, Fig. 1C and Fig. 3A
(lower panel).
The attachment and growth of the tissueoids was continuously monitored as a
function of time
using phase contrast microscopy. It was observed that the entire process was
completed in less than 24
hours or less than 48 hours or less than72 hours; including approximately 24
hrs to prepare the spheroids,
24 hrs to attach the spheroids on the base matrix system and a few hours for
spheroids to proliferate and
generate as tissueoids. After this, the AXTEX-4D system (cells growing on base
matrix system in 3D
culture) was ready to conduct screening studies and other analyses
demonstrated in other examples. In this
tissueoid base matrix system, spheroids of various primary cells and tissues,
pathological and non-
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pathological, cancer cells or patient tumor biopsies, transfected and non-
transfected cell lines were observed
to be grown with similar morphology to tissues in vivo.
Tissueoids was also generated from tumor biopsy by taking either suspension
culture and or explant.
Growth of cells of an explant inoculum to form a tissueoid was observed in
less than 24 hours or less than
48 hours or less than 72 hours of incubation in the 8% CO2 incubator. After
this, the platform was ready to
perform screening studies and other analyses.
Example 4: Types of materials of construction and thickness parameters for
base matrix system:
Different types of spun woven fabric materials, such as PET fabric with a
various density (19, 20,
30, 35) gm/m2 were used as 3D base matrix system. A representative example,
Fig. 2 shows efficient growth
of HT-29 tissueoids each on the same fabric and having different densities
(ranges from 19, 20, 30 and 35
gm/m2). Other materials that were also tested as base matrix system to grow
tissueoids included: FNT best
bond PP/PS/PA-40g/m22; FNT Cisellina PET 250g/m2; FNT Newjet viscose 80g/m2;
FNT Polibond PP
45g/m2; Hydroweb BicoPET/PP 150g/m2; JM 011/120 PET /120g/m2; Mogul Buffalo
bico PET/coPET,
round 80g/m2; Mogul Buffalo bico PET/coPET, tiptrilobal 80g/m2; Mogul Mopet
PET flatbond 19g/m2;
Mogul Mopet PET flatbond 75g/m2; Resintex Master PE, acrylic resin 220g/m2;
AS10; AS03; and ASO3A.
Example 5: Cell-extra cellular matrix interaction: Cell-extra cellular matrix
interaction plays an important
role for the tumor growth and invasion and serves as a crucial component of
tumor microenvironment.
Presence of collagen as ECM component involves in cancer fibrosis. Collagen in
presence of other
components like hyaluronic acid, fibronectin, laminin and matrix
metalloprotease influences cancer cell
activity. Tissuoids generated from MCF-7 cell line, grown on AXTEX-4D system,
were observed to
produce collagen (Fig. 7). ECM formation was more contiguous in case of
tissueoids grown on AXTEX-
4D as compared to the 2D monolayer cultures.
Example 6: Analysis of 2D and 3D cell culture sensitivity to drugs
The MCF-7 cell line was grown as 2D monolayer cultures as well as tissueoids
grown on 96 well
plate, where the spheroids were cultured on top of the membrane and incubated
for 1 to 3 days. A cell
number of approximately 5x 103 cells per spheroid were added to each well in
96 well plate either pre-
coated with 1.5% agarose in tissue culture coated 96 well plate or without
agarose in tissue culture uncoated
96 well plate. After attachment of spheroids, media was replaced with fresh
media in presence and absence
of drugs. In 2D culture 5x103 cells were seeded in each well of a 96 well
plate. Drug treatment was initiated
after attachment of cells for 48-72 hrs.
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As shown in Fig. 10, MCF-7 cells grown in 2D culture or on AXTEX-4D base
matrix system, were
treated with doxorubicin (1-5 M), the cells growing in 3D format showed
greater resistance to growth
arrest or killing compared to the cells cultured as 2D monolayer (Fig. 10).
Fig. 10A shows effect of
doxorubicin in different concentrations (1-5 M) on cell viability as they are
grown in a 2D monolayer, and
Fig. 10B shows effect of doxorubicin in different concentration (1-5 M) on
cell viability as grown as a 3D
tissueoid on AXTEX-4D base matrix system. As shown in Fig. 11A, cells and
tissueoids were treated with
different doses (1-5 M) of doxorubicin and partial resistance was observed in
tissueoids with 1 M
doxorubicin compared to vehicle control.
Fig. 11B shows combined effect of both doxorubicin and bevacizumab on growth
of tissueoids of MCF-7.
The MCF-7 tissueoids, grown on AXTEX-4D base matrix system, were initially
serum starved for 5 hrs
and treated with either 10Ong/m1 VEGF-165 (Vascular endothelial growth factor-
165, which is a splice
variant or isoform, Cat No. 293-VE-010, R&D systems) alone or in presence of 1
M doxorubicin and
25 g/m1 of bevacizumab (sourced from Roche, 100mg/4 ml) for 6 days at 37 C and
8% CO2. Viability was
assessed by using prestoblue. The drug susceptibility was analysed by
fluorescence based studies
(Excitation 485 nm/Emission 595 nm) using prestoblue.
Tissueoids grown on AXTEX-4D base matrix system showed greater efficacy (about
57%) to prevent cell
growth even at 1uA4 doxorubicin in presence of bevacizumab at 25 g/ml.
Example 7. Evaluation of the tissueoids grown on AXTEX-4D base matrix system
by fluorescence
microscopy:
As a process for conducting immunofluorescence analysis, samples were fixed
with 4% PFA for
15 mins and was washed with PBS for 3 times, 5 mins each. Samples were
permeabilized with 0.1% triton-
x. Staining was performed with anti-collagen type-I antibody (green) in 1:50
dilution, DAPI (blue- nucleus
stain) on Day 7 and observed under fluorescence microscopy. The ECM formation
in the tissueoids formed
on AXTEX-4D system was observed to be more contiguous as seen in images (Fig.
7A-Fig. 7F) as
compared to that of cells cultured in 2D monolayer. (Magnification 10X). The
samples were analyzed using
ApoTome microscope.
Example 8. Confocal analysis
Growth of tissueoids in 3D was visualized using confocal microscopy. For
performing confocal
analysis, the samples were fixed, stained and analysed using Leica TCS 5P8. In
this example, spheroids of
MCF-7 cells were prepared (as described herein) and added on the top of the
membrane, and incubated to
obtain growth of tissueoids.
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Cells were stained for F-actin using phalloidin and for nuclear staining using
Hoechst dye. Cells
were fixed with the fixatives and blocked for 30 mins at RT in PBS with 1%
BSA. Afterwards, the
specimens were washed in PBS, stained for actin followed by counter-staining
with Hoechst for nucleus
visualisation. Phalloidin staining was done using 1:1000 dilution for 40 mins
at 25 C. Nuclear staining was
performed using Hoechst at 1:1000 dilution in PBS for 15 minutes at 25 C (Fig.
8). Photographs were
taken at 10X magnification for 3D and 40Xmagnification for 2D using Leica
confocal microscopy (Leica
SP8).
To analyse the expression of collagen in tissueoids, cells were fixed and
blocked as described earlier.
Tissueoids of MCF-7 cell lines were stained with anti ¨collagen I antibody in
1:50 dilution for 16 hours.
Nucleus staining was performed using DAPI. Photographs were taken at same
magnification described
before.
To analyse the proliferation of longevity of tissueoids derived from PC3 cell
line, tissueoids were stained
with calcein AM for 30 minutes as per manufacturer's protocol. As shown in
Fig. 6, tissueoids generated
from PC3 cell line grown on base matrix AXTEX-4D are viable and able to
proliferate upto 250 days.
To analyse proliferation and viability of PC3 tissueoids, calcein AM (Thermo
Fisher) staining was
performed at different time points (day 3, day 25, day 108 and day 250). PC3
tissueoids were stained with
1[IM of calcein AM for 30 minutes and kept at 37 C and 8% CO2. Then tissueoids
were analysed by
confocal microscopy, the results showing increase in cell number viability as
shown by Calcein AM
staining (Fig.6)
Confocal analysis data showed tissue like organization of tissueoids of the
MCF-7 cell line with
contiguity of the cells clearly visible. This is in contrast to the picture
seen from the cells grown in Petri
dish as a 2D format in which the cells have defined edges and margins and are
non-contiguous (Fig. 8).
Example 9: Small cell numbers of initial sample as inocula for growth of
tissueoid on AXTEX-4D
Data showed that as few as 25 cells successfully grew and formed a tissueoid
on the base matrix
system. Analysis was done by making cell suspension by dilution method such
that 20 ttl of the media
contained a precise number of cells, ranging from 25 to 250 cells. The drop
was pipetted onto the inner
surface of a lid filled with PBS at the bottom. After 24 hrs, the inner lid
was inverted and the drops were
re-suspended in a fresh media. Spheroids were analyzed by phase contrast
microscopy and were added on
the top of base matrix placed in a tissue culture plate (24 or 96 well plates)
and incubated in a humidified
incubator at 37 C and 8% CO2. The attachment and growth of the tissueoids
using different inoculating
numbers of cells was investigated under phase contrast and scanning electron
microscope (Fig 9).
As illustrated in the figure, different cell number of MCF-7 cell line were
used as starting material
to grow spheroids and 3D tissueoids, ranging from about 250 cells to as few as
less than 25 cells:. Spheroids
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were grown on the 3D base matrix system as shown in Figs. Fig. 9A, Fig. 9B,
Fig. 9C, Fig. 9D, and Fig.
9E using phase contrast microscope (10X magnification) and Fig. 9F using SEM
analysis (1500X
magnification). The photographs show growth of tissueoids and clearly showed
that as few as 25 cells were
needed to create a tissueoid on the base matrix system provided herein.
Example 10: Viability time course of cell cultures on base matrix system and
culture duration
Growth and viability of tissueoids grown on base matrix, AXTEX-4D was observed
to have
extended to more than 1 year (approximately 364 days, Fig. 13, lower panel)
for PC3 and approximately
3 months for HepG2 (-82 days, Fig. 13, upper panel) and close to 130 days for
MCF-7 tissueoids ( We
have analysed and observed this phenomenon with three different cell lines
(HepG2, MCF-7 and PC3) on
the AXTEX-4D base matrix system base matrix system (19 gm/m2).
Further, viability of PC3 tissueoid was analysed at day 100 of its growth by
FACS analysis using
LIVE/DEAD stain and it was found that out of gated population i.e. (-75% ),
47. % cells are live and
18.68% cells are dead suggesting almost 60% viability of PC3 tissueoids even
after 100 days of culture.
Distinct advantages of prolonged growth and viability is provided by data
herein showing ability
to mimic tissue like conditions ex vivo for a longer period so that different
assays are performed for an
extended period of time, as a method to obtain the drug sensitivity data to
design the best therapeutic
regimen for a patient.
Example 11: A plurality of different cell lines were grown on the base matrix
system:
The tissueoid generation methods and systems described in this application are
a universal base
matrix system that was shown in examples herein capable of use for culture of
different types of cells in a
3D format. The following cell lines were successfully grown on the fabric base
matrix systems using the
process described above in Example 2 (, Fig. 1B, Fig. 1C, Fig, 2, Fig. 3Aõ
Fig. 4,) Cancerous cell lines:
MCF-7: breast cancer cell line; adenocarcinoma; HepG2: liver carcinoma;
epithelial cells; PC3: prostate
cancer cell line; adenocarcinoma; A375: skin melanoma; epithelial cell line;
HT-29: colorectal;
adenocarcinoma and non-malignant cell line CHO-Kl cells (stably expressing
surface protein); HEK-293;
NIH3T3 Fibroblasts. This system has also been tested for growth of tissueoids
derived from primary tumor
tissues like colon, gastric, lung and thyroid (represented in Fig. 3B and,
Fig. 5).
Example 12: Co-culture and tri-culture of different cell mixtures on the base
matrix system
Tissueoids were generated from mixed cell populations by co-culturing two or
more cell lines. Co-
culture of each of the combinations were analysed by taking either breast
cancer cell line (MCF-7) and
endothelial cells (HUVEC) or endothelial cells (HUVEC) and fibroblast (NIH-
3T3) in 1:1 ratio
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respectively. These were grown in 2D monolayer format and on the AXTEX-4D base
matrix system, as
shown in Fig. 14. The tissueoids were seen to grow very efficiently.
Tissueoids were generated of mixed cell populations by co-culturing three cell
lines. The cell
suspension of MCF-7 cell line was mixed with NIH-3T3 and HUVEC cell line in
1:2:1 ratios. These mixed
cell populations were grown on the AXTEX-4D and the data is shown in Fig. 14,
indicating that the
populations grew very efficiently.
Producing and analyzing co-cultures and tri-cultures using the tissueoid base
matrix system was
envisioned as useful in studying cell-cell interaction, drug discovery and
development and also for patient
treatment regimen, especially for the immune-oncology and infectious disease
base matrix systems Figs.
14.
Example 13: Primary cells and tissue samples from patients grown on the AXTEX-
4D base matrix system
Primary cell lines and samples from tissue biopsies from oncology patients
were grown on the base matrix
system as tissueoids. Tumor tissue specimens were taken as suspension culture
or as explant and grown on
AXTEX-4D base matrix system. The growth of tumor tissue on the base matrix
system is shown as an
example in Fig. 3B and Fig. 5 demonstrating that AXTEX-4D base matrix system
can be effectively and
universally used to generate tissueoids from primary tissue samples/ biopsies.
Example 14: Reduction in time to grow the tissueoid on the base matrix system
and initiate the assays
Observations herein report a time interval equal to or less than 24 hrs for
the cells from cell lines/
primary cells to attach to the AXTEX-4D base matrix system and initiate
growing as tissueoids. The
tissueoid was observed to be suitable for analyses for drug sensitivity and
resistance appropriate to
therapeutic drug regimen assays. In certain cell lines, it was observed that
the cells required somewhat more
time, but generally for human cell lines no more time than 72 hours was
required for the attachment on the
base matrix system and to start growth as a tissueoid. This rapidity of
culture of tissueoids addresses a long
felt need and the key critical factor for any patient-drug related studies
that is factor of time and makes it a
four-dimensional system. (Fig. 15).
Example 15: Cell factory
AXTEX-4D base matrix system sustains the growth of tissueoid for longer time
duration. It is
envisioned that for large-scale production of cells, vaccines, and therapeutic
proteins, antibodies, secretory
proteins the 3D systems and methods and format provided herein are very
useful. The system is convenient
to handle, requires no special tubing and increased antibody production was
observed as a function of time
as is shown in Fig. 16.
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CHO-DG44 cells stably expressing tocilizumab, an antiIL-6R antibody, growing
on AXTEX-4D
base matrix system allowed increased number of cells in a more compact space
with increased longevity
and better productivity of the antibody expressing cells (Fig. 16). This
confirms use of AXTEX-4D base
matrix system for cell factory for biotherapeutic production of biologics and
vaccines.
Example 16: Angiogenesis base matrix system
Endothelial cell dysfunction has a role in diabetes, pulmonary diseases,
inflammatory diseases,
cardiovascular diseases and immune diseases etc. Angiogenesis is a critical
process for tissue development,
wound healing and tumor progression. The methods utilising 3D format provided
useful insights for
studying angiogenesis or tumor microenvironment screening for inhibitors of
anti-angiogenic drug.
Tissueoids generated from loosely compact HUVEC cells were grown on AXTEX-4D
system in presence
of VEGF-165, which is a potent mediator of angiogenesis. Fig. 17 represents
growth of tissueoids along
with tube like structure formation after treatment with 50 ng/ml of VEGF
treatment for 72 hrs.
The 3D methods and systems provided herein have yielded important insights
into angiogenesis and
creation of the tumor microenvironment and the need for screening potential
anti-angiogenesis drugs in a
system that closely resembles that of a tumor in vivo.
