NZ794605A - Neural tissue unit and use of such a unit for implantation in the nervous system of a mammal - Google Patents
Neural tissue unit and use of such a unit for implantation in the nervous system of a mammalInfo
- Publication number
- NZ794605A NZ794605A NZ794605A NZ79460517A NZ794605A NZ 794605 A NZ794605 A NZ 794605A NZ 794605 A NZ794605 A NZ 794605A NZ 79460517 A NZ79460517 A NZ 79460517A NZ 794605 A NZ794605 A NZ 794605A
- Authority
- NZ
- New Zealand
- Prior art keywords
- cells
- cellular
- microcompartment
- cell
- preferentially
- Prior art date
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Abstract
The invention relates to a neural tissue unit for use in implantation into the nervous system of a human or non–human mammal, wherein said neural tissue unit contains differentiated post–mitotic neuronal cells in an extracellular matrix, said unit being obtained from a cellular microcompartment comprising a hydrogel capsule surrounding the neural tissue unit, and said hydrogel capsule being at least partially removed before use of the neural tissue unit. The invention also relates to a process for preparing such a neural tissue unit. rising a hydrogel capsule surrounding the neural tissue unit, and said hydrogel capsule being at least partially removed before use of the neural tissue unit. The invention also relates to a process for preparing such a neural tissue unit.
Description
The invention relates to a neural tissue unit for use in implantation into the nervous system
of a human or non–human mammal, wherein said neural tissue unit contains differentiated
itotic neuronal cells in an extracellular matrix, said unit being obtained from a cellular
microcompartment comprising a hydrogel capsule surrounding the neural tissue unit, and
said hydrogel e being at least partially removed before use of the neural tissue unit. The
invention also relates to a process for preparing such a neural tissue unit.
NZ 794605
ar microcompartment and preparation methods
This application is a divisional of New d ation No. 752888, filed 23 November
2017, and claims the benefit of French Provisional Application No. 1661377, filed
23 November 2016, all of which are incorporated herein by reference in their entirety.
The ion relates to a cellular microcompartment allowing the pluripotency of human cells
to be maintained. The invention also relates to a ation process for obtaining such three–
dimensional (3D) cell culture compartments.
Pluripotent cells are considered an important human cell resource, and their cultivation is of
growing interest, particularly in the medical and pharmaceutical fields. Thus, producing
otent cells in large quantities would meet the new needs expressed by the pharmaceutical
industry, which is reducing use of animal models in favor of cell models which are more
relevant than the many cell lines tly in use. High throughput tests ped by
pharmaceutical companies already use large quantities of human pluripotent cells. rly,
tissue engineering and cell therapy in humans rely on the availability of industrial quantities of
human pluripotent cells.
tly, human pluripotent cells are usually cultured in a two–dimensional (2D) environment,
such as on Petri dishes, very different from the 3D medium in which the cells normally evolve.
The manipulation of these 2D ed cells is often delicate, and requires in particular steps of
purification, enzymatic detachment, etc. Furthermore, these cells are difficult to store and have
a very low survival rate after freezing. However, the ability to use conventional rs to send
pluripotent cell cultures frozen in massive quantities, and compatible with liquid culture,
represents a major challenge for both research laboratories and pharmaceutical industries.
In response to this ion, three–dimensional culture systems, which seek in particular to
increase the throughput, efficiency and quality of human pluripotent stem cell culture systems,
have been developed.
r, ng 3D culture systems are not entirely satisfactory. Uncontrolled fusion
phenomena are often observed, resulting in cell aggregates whose size (>200 µm in diameter)
makes the diffusion of the culture medium insufficient. Thus, within such 3D culture systems,
cell differentiation is difficult to control and/or the cell death rate is very high. Generally, the
lack of homogeneity of products derived from 3D cell culture and the cost of such techniques
make this technology uncompetitive compared with 2D culture, which however is
unsatisfactory.
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There is thus a need for a 3D cell culture system that can provide large quantities of pluripotent
cells with a controlled phenotype, which can be easily used both for basic research and
industrially.
Summary of the invention
While g on the development of cellular ompartments for 3D cell culture, the
inventors developed a system that allows mass liquid suspension culture of human pluripotent
cells while maintaining their phenotype. The developed microcompartments allow cells to be
cultured in liquid medium, using the media conventionally used in 2D culture, while protecting
the cells and controlling their phenotype to avoid ype differentiation and maintain
pluripotency. More precisely, the microcompartments, or es, developed by the inventors
comprise successively, organized in a substantially homocentric , a hydrogel shell, an
extracellular matrix layer and one or more layers of human pluripotent cells surrounding a
l lumen. The hydrogel shell of the capsules according to the invention, unlike existing
culture s, protects the cells from the mechanical stresses associated with collisions or
fusions during liquid suspension culture. Particularly advantageously, the organization in
“cysts” of the ompartments according to the invention allows them to be frozen with a
high cell survival rate. In addition, the cells can be differentiated before use, directly within the
ompartment, or used in the pluripotent stage, in both 3D and 2D culture. The inventors
have also developed methods for preparing such ar microcompartments, guaranteeing that
the cyst form is obtained and maintained, which are suitable both for freezing and for
controlling the phenotype of the cells they contain.
The t–matter of the invention is therefore a cellular microcompartment comprising
successively, organized around a lumen:
- at least one layer of human pluripotent cells;
- an extracellular matrix layer;
- an outer hydrogel layer.
Advantageously, culture medium fills the spaces left between the layers.
Another subject–matter of the invention is a process for ing a cellular microcompartment
according to the invention, comprising the steps consisting in
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(a) ting human pluripotent stem cells in a culture medium containing a RHO/ROCK
pathway inhibitor;
(b) mixing the pluripotent stem cells from step (a) with an extracellular matrix;
(c) encapsulating the mixture from step (b) in a hydrogel layer;
(d) culturing the capsules obtained in step (c) in a culture medium containing a RHO/ROCK
y tor;
(e) rinsing the capsules from step (d) to remove the RHO/ROCK pathway inhibitor;
(f) culturing the capsules from step (e) for 3 to 20 days, preferentially 5 to 10 days, in a culture
medium free of RHO/ROCK pathway inhibitor, and optionally recovering the ar
microcompartments obtained.
Another subject–matter of the invention is a process for preparing a cellular microcompartment
according to the invention, comprising the steps ting in
(a) mixing human differentiated cells with an extracellular matrix and cell reprogramming
agents;
(b) encapsulating the mixture from step (a) in a hydrogel layer;
(c) culturing the capsules from step (b) for 10 to 40 days, and optionally recovering the cellular
microcompartments obtained.
Brief description of the figures
Figure 1: Photo (A) and schematic representation (B) of a ar microcompartment forming
a cyst according to the invention (1: hydrogel layer; 2: ellular matrix layer; 3: layers of
pluripotent cells; 4: lumen).
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Detailed description
The subject–matter of the invention is a 3D cellular microcompartment comprising human
pluripotent cells, in which the pluripotency of the cells is maintained.
Cellular ompartment
The cellular ompartment according to the invention forms a cyst whose hollow center,
or lumen, is preferentially aqueous. In the context of the invention, a “cyst” refers to a closed
hollow structure containing substantially homocentric layers, in the sense that they are
organized successively around the same point, the outer layer enveloping the matrix layer which
envelops the cell layer, which surrounds the lumen. Generally, the otent cells making up
the cyst are polarized. The polarity of these cells within the cyst can be detected by the proteins
TJP–1 or ZO–1, both located on the inner/apical side of the pluripotent cell layer adjacent to
the lumen.
The lumen is generated, at the time of cyst formation, by the cells that multiply and develop on
the extracellular matrix layer. Advantageously, the lumen contains a liquid and more
ularly culture medium.
In the context of the invention, the “hydrogel layer” refers to a three–dimensional structure
formed from a matrix of r chains swollen by a liquid, preferentially water.
ageously, the hydrogel used is biocompatible, in the sense that it is not toxic to cells.
Furthermore, the hydrogel layer must allow the diffusion of oxygen and nutrients to feed the
cells contained in the microcompartment and allow them to survive. For example, the outer
hydrogel layer contains alginate. Preferentially, the outer layer contains only alginate. In the
t of the invention, “alginate” refers to linear polysaccharides formed from β–D–
mannuronate (M) and α–L–guluronate (G), salts and derivatives f. Advantageously, the
alginate is a sodium alginate, composed of more than 80% G and less than 20% M, with an
average molecular mass of 100 to 400 KDa (e.g., PRONOVA® SLG100) and a total
concentration comprised between 0.5% and 5% by mass. According to the invention, the
hydrogel layer is cell–free. In one embodiment of the cellular ompartment according to
the invention, the outer layer comprises alginate.
In turn, the extracellular matrix layer may n a few cells. Indeed, at the time of cyst
formation, the cells create their space in the matrix and multiply, filling the ompartment.
The boundary between the extracellular matrix layer and the pluripotent cell layer may therefore
not be perfectly clear. At the surface in contact with the cell layer, the extracellular matrix may
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thus contain a few pluripotent cells. Conversely, the surface of the extracellular matrix layer in
contact with the hydrogel layer is cell–free.
The extracellular matrix layer is necessary for the survival of pluripotent cells in the
microcompartment and for the on of the cyst.
Preferentially, the ellular matrix layer forms a gel on the inner side of the hydrogel layer,
meaning the side directed towards the lumen of the microcompartment. The extracellular matrix
layer ses of a mixture of proteins and extracellular nds necessary for cell culture,
and more particularly the culture of pluripotent cells. Prefer entially, the extracellular matrix
comprises structural proteins, such as laminins containing α1, α4 or α5 subunits, β1 or β2
subunits, and γ1 or γ3 subunits, entactin, vitronectin, laminins, collagen, as well as growth
s such as TGF–beta and/or EGF. In one embodiment, the extracellular matrix layer
consists of, or contains, Matrigel® and/or Geltrex®.
According to the invention, the cellular microcompartment contains one or more layers of
human otent stem cells. A pluripotent stem cell, or pluripotent cell, is a cell that has the
ability to form all the s present in the whole original organism but cannot form a whole
sm as such.
In a particular embodiment, the encapsulated cells are otent stem cells, such as induced
pluripotent stem (IPS) cells, multilineage–differentiating stress ng (MUSE) cells found
in the skin and bone marrow of adult mammals, or embryonic stem (ES) cells.
In the context of the invention, “induced otent stem cells” (IPS cells) are d as
pluripotent stem cells obtained by genetic reprogramming of differentiated somatic cells and
having a morphology and a potential for self–renewal and pluripotency partially similar to those
of embryonic stem cells. These cells are notably ve for pluripotency markers, such as
alkaline phosphatase staining and expression of the proteins NANOG, SOX2, OCT4 and
SSEA3/4. The processes for obtaining induced pluripotent stem cells are well known to the
skilled person and are notably described in articles by Yu et al. (Science, 2007, 318 (5858):
1917–1920), Takahashi et al. (Cell, 2007, 131(5): 861–872) and Nakagawa et al. (Nat
Biotechnol, 2008, 26(1): 101–106).
In the case of embryonic stem cells, said pluripotent stem cells are cells derived from the
internal cell mass of the blastocyst and which have the ability to lead to the formation of all
tissues of the organism. The pluripotency of nic stem cells ca n be assessed by the
presence of markers such as the transcription factors OCT4 and NANOG and surface markers
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such as SSEA3/4, Tra–1–60 and 81. Embryonic stem cells can be obtained without
destroying the embryo from which they originate, for example by using the technique described
by Chung et al. (Cell Stem Cell, 2008, 2(2): 113–117). In a particular embodiment, and for
legal or ethical reasons, stem cells are defined as excluding human embryonic stem cells.
In one embodiment, the human pluripotent stem cells used for the microcompartments
according to the invention are induced to pluripotency from somatic cells.
Advantageously, the cell layer contains at least 95% by volume, preferentially at least 96%,
97%, 98%, 99% of cells and of matrix produced by said cells. The cells are essentially
pluripotent cells. “Essentially” means that at least 90% of the cells contained in the cell layer
are pluripotent cells, preferentially at least 95%, 96%, 97%, 98%, 99%, 100%, are pluripotent
cells.
Advantageously, the lumen of the cyst contains culture . In particular, any culture
medium ng the suspension culture of pluripotent cells may be used, and in ular any
culture medium tionally used in 2D culture.
Preferentially, the cellular microcompartment is closed. It is the outer hydrogel layer that gives
the cellular microcompartment its size and shape. The microcompartment can have any shape
compatible with cell encapsulation.
Advantageously, the dimensions of the cellular ompartment are controlled. In one
embodiment, the cellular microcompartment according to the invention has a spherical shape.
Preferentially, the er of such a microcompartment is comprised between 10 µm and
1 mm, more preferentially between 50 µm and 500 µm, even more preferentially is less than
500 µm, ably less than 400 µm.
In another embodiment, the cellular microcompartment ing to the invention has an
elongated shape. In particular, the microcompartment may have an ovoid or r shape.
Advantageously, the smallest dimension of such an ovoid or tubular microcompartment is
comprised between 10 µm and 1 mm, more preferentially between 50 µm and 500 µm, even
more preferentially less than 500 µm, preferentially less than 400 µm. “Smallest dimension”
means twice the minimum distance between a point on the outer surface of the hydrogel layer
and the center of the microcompartment.
In a particular embodiment, the thickness of the outer hydrogel layer represents 5 to 40% of the
radius of the microcompartment. The ess of the ellular matrix layer represents 5 to
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80% of the radius of the microcompartment and is advantageously attached to the inner side of
the hydrogel shell. The thickness of the otent cell layer ents about 10% of the radius
of the microcompartment. The pluripotent cell layer is in t at least at one point with the
extracellular matrix layer, a space filled with culture medium may be present between the
matrix layer and the cyst. The lumen then represents 5 to 30% of the radius of the
microcompartment. In the context of the invention, the “thickness” of a layer is the dimension
of said layer extending radially relative to the center of the microcompartment.
In a particular example, the ar ompartment has a spherical shape with a radius of
100 µm. The hydrogel layer has a thickness of 5 µm to 40 µm. The extracellular matrix layer
has a thickness of 5 µm to about 80 µm. The layer of pluripotent cells has a thickness of 10 to
µm, the lumen has a radius of 5 to 30 µm, roughly.
In l, the presence of the outer hydrogel layer imposes a maximum size on the cell layer
and limits, by confinement, the uncontrolled proliferation of cells, which could lead to the
anoxic death of the cells and/or uncontrolled differentiation of the cells in the deepest layers,
meaning those closest to the lumen of the cyst. In 2D, on a Petri dish, the colonies are discs, the
cells at the center of the disc tend to die (each new cell resulting from a division is excluded
from the colony by the lack of space) or to differentiate under the constraints of the cells
surrounding them, the cells on the edge tend to differentiate and only a band at the right distance
has the optimal phenotype. The topology of the ompartment ted here, the inner
surface of the sphere formed by the capsule, makes it possible to generate a “colony” of stem
cells (the pluripotent cell layer) “without edges” where all the cells are optimally and equally
positioned both for the ion of small molecules and in terms of mechanical stresses.
Advantageously, the cell density in the microcompartment is comprised between 1 and several
thousand cells per microcompartment, entially between 50 and 1000 cells per 100 µm
radius microcompartment.
Processes for preparing cellular microcompartments
The invention also relates to processes for preparing cellular microcompartments which make
it possible to obtain the cellular microcompartment according to the invention. More
specifically, the invention proposes to produce cellular microcompartments containing
pluripotent stem cells zed into cysts directly from pluripotent stem cells, or from
differentiated cells which will be reprogrammed into otent cells inside the hydrogel
capsule during the formation of the microcompartments.
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Any method for producing cellular microcompartments containing extracellular matrix and
pluripotent stem cells within a hydrogel capsule may be used for the implementation of the
preparation process according to the invention. In particular, it is possible to prepare
microcompartments by adapting the microfluidic method and device described in Alessandri et
al., 2016 (“A 3D printed microfluidic device for production of functionalized hydrogel
microcapsules for culture and differentiation of human Neuronal Stem Cells ”, Lab on
a Chip, 2016, vol. 16, no. 9, p. 1593–1604), in accordance with the steps described below.
In a first embodiment, the preparation process according to the invention comprises the steps
ting in
(a) incubating human pluripotent stem cells in a culture medium containing a RHO/ROCK
pathway inhibitor;
(b) mixing the pluripotent stem cells from step (a) with an extracellular matrix;
(c) encapsulating the mixture from step (b) in a hydrogel layer;
(d) culturing the capsules obtained in step (c) in a culture medium ning a RHO/ROCK
pathway inhibitor;
(e) rinsing the capsules from step (d) to remove the RHO/ROCK y inhibitor;
(f) culturing the capsules from step (e) for 3 to 20 days, entially 5 to 10 days, until a cyst
is obtained, and optionally recovering the ar microcompartments ed.
Incubation step (a) and culture step (d) in a medium containing one or more RHO/ROCK
(“Rho–associated protein kinase”) pathway inhibitors, such as vivin (C15H13N5OS)
and/or Y–27632 (C14H21N3O), promote the survival of pluripotent stem cells and cell adherence
to the extracellular matrix when the outer hydrogel layer is formed around said extracellular
matrix. It is however desirable that these steps be limited in time, so that RHO/ROCK pathway
inhibitors do not prevent the formation of cysts.
Thus, preferentially, the incubation of step (a) is conducted for a time comprised between a few
minutes and a few hours, preferentially between 2 s and 2 hours, more entially
between 10 minutes and 1 hour.
(40873282_1):CLVRH
Similarly, preferentially, culture step (d) is conducted for a time comprised between 2 and 48
hours, preferentially for a time comprised n 6 and 24 hours, more entially for a
time comprised between 12 and 18 hours.
Step (e) is necessary to ensure the removal of all traces of RHO/ROCK pathway inhibitors. Step
(e) is carried out, for example, by g, and preferentially by several rinses, in successive
culture media free of RHO/ROCK pathway inhibitors.
Advantageously, step (f) is conducted for a sufficient time to obtain a cellular
microcompartment in which the pluripotent cell layer and the lumen have a tive
thickness equal to 10 to 95% of the radius of the microcompartment, that is to say, for a
sufficient time to allow to pass from two cells to about a thousand cells. Any culture medium
suitable for pluripotent stem cell culture may be used, and notably saline ate buffer such
as Roswell Park Memorial Institute medium.
In one embodiment, the s according to the invention comprises an intermediate step (a′)
consisting in dissociating the pluripotent stem cells from step (a) before step (b), preferentially
by means of an enzyme–free reagent. ageously, said reagent is inhibited or rinsed before
the encapsulation step, in particular by successive rinses in a specific medium for pluripotent
cells. For example, the reagent used is an iso–osmotic buffer ning EDTA or EGTA such
as ReLeSR®. Of course, it is also le to use trypsin or a reagent containing an ,
but the survival rate of pluripotent cells at the end of this step may then be lower compared with
the use of an enzyme–free reagent. In all cases, the rinsing step is necessary to remove any trace
of the reagent used for cell dissociation.
In one embodiment, at least one of steps (a′), (b), (c), (d) or (e) is performed at a ature
comprised between 0 and 8°C, preferentially all of steps (a′), (b), (c), (d) and (e). Maintaining
a temperature substantially equal to 4°C allows the biological processes of the cells to become
dormant, including the transduction of signals from the external environment. This makes it
possible to limit the phenomenon of cell death, which could be induced by cell detachment.
In another embodiment, the process for preparing a cellular microcompartment according to the
invention comprises the steps consisting in
(a) mixing differentiated human cells with an extracellular matrix and cell reprogramming
agents that do not permeate the hydrogel layer;
(b) encapsulating the mixture from step (a) in a hydrogel layer;
(40873282_1):CLVRH
(c) culturing the capsules from step (b) for 10 to 40 days, and optionally recovering the cellular
microcompartments obtained.
In another embodiment, the s for preparing a ar microcompartment according to the
invention ses the steps consisting in
(a) mixing differentiated human cells with an extracellular matrix;
(b) encapsulating the mixture from step (a) in a hydrogel layer;
(c) incubating the capsules from step (b) with ar ramming agents that permeate the
hydrogel layer and culturing the capsules for 10 to 40 days, and optionally recovering the
cellular microcompartments obtained.
For example, the differentiated cells used are lasts.
The skilled person knows how to reprogram a differentiated cell into a stem cell by reactivating
the expression of genes associated with the embryonic stage by means of specific s,
referred to in the present invention as “reprogramming agents”. Examples include the methods
described in Takahashi et al., 2006 (“Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors” Cell, 2006 Vol 126, pages 663–676), Ban et
al., 2009 (“Efficient ion of transgene–free human pluripotent stem cells using a vector
based on Sendai virus, an RNA virus that does not integrate into the host genome” Proc Jpn
Acad Ser B Phys Biol Sci. 2009; 85(8):348–62) and in international application
WO2010/105311 entitled “Production of reprogrammed otent cells”.
The reprogramming agents are advantageously apsulated with the differentiated cells,
so as to concentrate the product and promote contact with all the cells. In the case of
reprogramming agents that permeate the hydrogel layer, it is possible to add said agents to the
culture medium after the encapsulation step.
The reprogramming agents make it possible to impose on the cells a succession of phenotypic
changes up to the pluripotent stage. Advantageously, reprogramming step (a) is med
using specific culture media, promoting these phenotypic changes. For example, the cells are
cultured in a first medium comprising 10% human or bovine serum, in Eagle’s minimal
essential medium (DMEM) supplemented with a serine/threonine protein kinase receptor
inhibitor (such as the product SB–431542 (C22H16N4O3)), one or more RHO/ROCK (“Rho–
associated protein ”) pathway inhibitors such as thiazovivin and/or Y–27632, fibroblast
growth factors such as FGF–2, ascorbic acid, and otics such as Trichostatin A
(40873282_1):CLVRH
(C17H22N2O3). Then the e medium is replaced by medium that promotes the multiplication
of pluripotent cells, such as 1.
ageously, the capsules from step (b) each contain between 1 and 500 differentiated cells,
preferentially between 50 and 200.
In one embodiment, at least one of steps (a), (b), (c) or (d) is performed at a temperature
comprised between 0 and 4°C, preferentially all of steps (a), (b), (c) and (d). Maintaining a
temperature of 4°C or lower allows the biological processes of the cells to become dormant,
including the transduction of signals from the external environment. This makes it le to
limit the phenomenon of cell death, which could be induced by cell detachment.
The microcompartments obtained during step (d) can be sorted so as to isolate the cellular
microcompartments with the desired cyst form. Such a sorting step can be d out
continuously, so as to separate the cellular ompartments already having the desired cyst
form from the microcompartments still being formed. Such a sorting step can be done by simple
logical analysis, without disturbing the other microcompartments in which
reprogramming is still in progress and/or the cyst organization not yet completed.
In general, the cellular microcompartments ed by the processes of the invention may then
be frozen before use. Indeed, the cyst form promotes cell survival within the
microcompartment, and after thawing, the survival rate is greater than 80%. Advantageously,
freezing is carried out using liquid nitrogen to quickly vitrify the microcompartments and limit
the risk of crystal formation within the lipid membranes of the cells. The cellular
microcompartments may be suspended in a freezing buffer that promotes cell survival. For
example, it is possible to use the freezing s conventionally used to freeze embryos.
The cellular microcompartments, thus frozen, may then be thawed as needed.
Applications
The cellular microcompartments ned by the present invention can be used for many
applications. Indeed, the cells they contain can be easily recovered by simple hydrolysis and/or
dissolution of the outer hydrogel layer. rmore, it is possible to entiate pluripotent
cells within the hydrogel capsule or after hydrolysis/dissolution of said hydrogel capsule, as
needed, in order to obtain large quantities of cell lines of interest. Advantageously, the cells are
differentiated into one or more cell types of interest, within the microcompartment, meaning
before hydrolysis of the outer hydrogel layer.
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The cellular microcompartments, and more precisely the cells they contain, can be used for
research and development purposes, both in the form of a 3D cell network and more
conventionally in 2D e. They can also be used for therapeutic purposes, such as cell
therapy, tissue engineering, etc.
EXAMPLES
Example 1: Protocol for obtaining cellular ompartments from human cells induced
to pluripotency.
Solutions used:
Solution 1, DMEMF12 medium base supplemented with 2 µM Thiazovivin
Solution 2, PBS without magnesium and without calcium supplemented with 1 µM 2 µM
Thiazovivin
Solution 3, zymatic cell detachment buffer: RelesR™ supplemented with 2 µM
Thiazovivin.
on 4, pluripotent stem cell culture medium: MTeSR1™ hES/hIPS cell medium
STEMCELL™).
Solution 4+, Solution 4 supplemented with 2 µM Thiazovivin.
Solution 5, el™.
Solution 6, 300 mM sorbitol with 2 µM Thiazovivin.
Cell solution:
A 25 cm² Petri dish of human IPS cells (obtained from Primary Dermal Fibroblast; Normal,
Human, Adult ATCC® PCS–201–012™ and CytoTune™–iPS 2.0 Sendai Reprogramming Kit
(item number A16517) using the technology shown in example 2) at 90% confluence is then
used to match the recommended volumes. All the following steps are carried out at 4°C until
the hydrogel shell is crosslinked in the m bath.
Step 1: Rinse the cells with solution 1. Wait 10 s to 1 hour.
Step 2: Rinse twice with 4 mL of solution 2.
(40873282_1):CLVRH
Step 3: Gently aspirate the solution.
Step 4: Incubate the cells with 4 mL of solution 3 for 5–10 minutes.
Step 5: Detach the cells with 2 mL of solution 4+ with a wide–tipped pipette to reduce shear
stress.
Step 6: fuge the cell suspension at 360 g for 5 minutes.
Step 7: Aspirate the supernatant.
Step 8: Resuspend with 0.5 mL of solution 4+.
Step 9: Centrifuge again at 360 g and aspirate the supernatant.
Step 10: Resuspend the cell pellet in 70 µL of on 5 and 100 µL of solution 6 (the volume
of the pellet should be 30 µL). The cell solution is ready.
Encapsulation:
The encapsulation device is prepared as described in Alessandri et al., 2016 (“A 3D printed
microfluidic device for production of functionalized hydrogel microcapsules for culture and
differentiation of human Neuronal Stem Cells (hNSC)”, Lab on a Chip, 2016, vol. 16, no. 9,
pp. 1593–1604).
In summary, the different parts of the device are ized (by autoclave); the three necessary
solutions are loaded on three syringe pumps, i) te solution (PRONOVA®SLG100 at 2%
by mass in distilled water), ii) intermediate solution (300 mM ol), iii) cell solution
red in the previous step); the three ons are co–injected concentrically using a
microfluidic injector which forms a jet that breaks down into drops whose outer layer is the
alginate solution and the core the cell solution; these drops are collected in a calcium bath (at
100 mM) that stiffens the alginate solution to form the shell.
To improve the monodispersity of the cellular microcompartments, the alginate was charged
with a +2 kV DC current. A mass ring of 2 cm in diameter is placed 500 µm from the tip in the
plane perpendicular to the axis of the jet leaving the microfluidic injector to generate the electric
field.
(40873282_1):CLVRH
It should be noted that under these ulation conditions, the Matrigel® layer forms
neously.
Treatment after encapsulation:
Step 1: The capsules are collected with a 40 µm cell sieve and then after rinsing with solution
1 they are stored in a 75 cm² flask with 20 mL of solution 4+.
Step 2: The flask is kept for 12 h in the incubator at 37°C and 5% CO2.
Step 3: Change the medium for solution 4 to allow the formation of cysts.
Step 4: After 24 to 72 hours, cysts of a few dozen cells are formed in the capsules. The cellular
microcompartments are mature after 5 to 10 days.
Example 2: Protocol for obtaining cellular microcompartments from human fibroblasts.
Solutions used:
Solution 1, DMEMF12 medium base
Solution 2, PBS t magnesium without added calcium
Solution 3, trypsin EDTA cell detachment buffer
on 4, fibroblast culture medium: 10% human serum in a DMEM medium base
Solution 4+, Solution 4 supplemented with 2 µM Thiazovivin.
Solution 5, Matrigel™.
Solution 6, 300 mM sorbitol with 2 µM Thiazovivin.
Cell solution:
A 25 cm² Petri dish of human fibroblasts (Primary Dermal Fibroblast; Normal, Human, Adult
(ATCC® PCS–201–012®) with low confluence density is then used to match the recommended
volumes (1 to 2 million cells). All the ing steps are carried out at 4°C until the shell is
crosslinked in the calcium bath.
(40873282_1):CLVRH
Step 1: Rinse the cells with solution 2.
Step 2: Gently aspirate the solution.
Step 3: Incubate the cells with 4 mL of solution 3 for 5–10 minutes.
Step 4: Detach the cells with 2 mL of solution 4+ with a wide–tipped pipette to reduce shear
stress.
Step 6: Centrifuge the cell sion at 360 g for 5 minutes.
Step 7: Aspirate the supernatant.
Step 8: Resuspend with 0.5 mL of solution 4+.
Step 9: Centrifuge again at 360 g and aspirate the supernatant.
Step 10: Resuspend the cell pellet in 90 µL of solution 5 and 100 µL of solution 6 (the pellet
volume should be 10 µL).
Step 11: Add 1/10 of the contents of the une® −IPS 2.0 Sendai Reprogramming Kit”
(containing a reprogramming virus) provided for a 6–well plate. The cell solution is ready.
Encapsulation:
The encapsulation is performed in accordance with the protocol of example 1.
Treatment after encapsulation:
Step 1: The capsules are collected with a 40 µm cell sieve and then after rinsing with solution
1 they are stored in a 75 cm² flask with 20 mL of on 4+.
Step 2: The flask is kept for 24 h in the tor at 37°C and 5% CO2.
Step 3: Change the medium every day. Each capsule contains 1 to 10 fibroblasts at capsule
formation. The reprogramming virus has a transformation efficiency of about 0.2%. Most of
the capsules will therefore contain very few reprogrammed cells, if any. Cysts begin to form
after 15 to 40 days. The fibroblasts have an ted shape and do not form cysts. Thus, all
the cysts that are formed are formed of IPS cells.
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Claims (11)
1. Cellular microcompartment comprising successively, organized around a lumen: - at least one layer of human pluripotent cells; 5 - an extracellular matrix layer; - an outer hydrogel layer.
2. Cellular ompartment ing to claim 1, wherein said microcompartment is closed.
3. Cellular microcompartment according to claim 1 or 2, wherein the outer layer 10 comprises te.
4. Cellular microcompartment according to one of the preceding claims, wherein said microcompartment has a spherical or elongated shape.
5. ar microcompartment according to one of the preceding claims, n said microcompartment has a diameter or a st dimension comprised between 10 µm and 15 1 mm, preferentially n 50 µm and 500 µm, more preferentially less than 500 µm, even more preferentially less than 400 µm.
6. Cellular microcompartment according to one of the previous , wherein the cell density is comprised between one and several thousand cells, preferentially 50 to 1000 cells per microcompartment. 20
7. Process for preparing a cellular microcompartment according to one of claims 1 to 6, comprising the steps consisting in: (a) incubating human pluripotent stem cells in a culture medium containing a RHO/ROCK pathway inhibitor; (b) mixing the pluripotent stem cells from step (a) with an extracellular matrix; 25 (c) encapsulating the mixture from step (b) in a hydrogel layer; (d) culturing the capsules obtained in step (c) in a culture medium containing a RHO/ROCK pathway inhibitor; (e) rinsing the es from step (d) to remove the CK pathway inhibitor; (f) culturing the capsules from step (e) for 3 to 20 days, preferentially 5 to 10 days, and 30 optionally recovering the cellular microcompartments obtained. (40873282_1):CLVRH
8. Process for preparing a microcompartment according to claim 7, comprising an intermediate step consisting in (a′) dissociating the pluripotent stem cells from step (a) before step (b), preferentially by means of an enzyme–free reagent. 5
9. Process for preparing a cellular microcompartment according to one of claims 1 to 6, comprising the steps consisting in (a) mixing human differentiated cells with an extracellular matrix and cell reprogramming agents; (b) ulating the mixture from step (a) in a hydrogel layer; 10 (c) culturing the capsules from step (b) for 10 to 40 days, and optionally recovering the cellular microcompartments obtained.
10. Process for preparing a cellular ompartment according to claim 9, n each capsule from step (b) contains between 1 and 500 differentiated cells.
11. Process for preparing a cellular ompartment according to one of claims 7 to 15 10, comprising a subsequent step consisting in freezing the cellular microcompartments ed in step (f) according to claim 7 or in step (c) according to claim 9. (40873282_1):CLVRH
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR1661378 | 2016-11-23 |
Publications (1)
Publication Number | Publication Date |
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NZ794605A true NZ794605A (en) | 2022-11-25 |
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