U.S. patent application number 17/546489 was filed with the patent office on 2022-06-30 for hydrogel compositions comprising encapsulated cells and methods of use thereof.
The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Angelo S. Mao, David J. Mooney, Jae-Won Shin, Stefanie Utech, Oktay R. Uzun, David A. Weitz.
Application Number | 20220202727 17/546489 |
Document ID | / |
Family ID | 1000006211410 |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220202727 |
Kind Code |
A1 |
Shin; Jae-Won ; et
al. |
June 30, 2022 |
HYDROGEL COMPOSITIONS COMPRISING ENCAPSULATED CELLS AND METHODS OF
USE THEREOF
Abstract
The present invention provides injectable compositions
comprising cells encapsulated in hydrogel capsules and methods of
preparing these compostions. The present invention also provides
methods for using these compositions to promote hematopoiesis and
to treat or prevent cardiovascular and immunological disorders in a
subject.
Inventors: |
Shin; Jae-Won; (Cambridge,
MA) ; Mao; Angelo S.; (Cambridge, MA) ; Utech;
Stefanie; (Cambridge, MA) ; Weitz; David A.;
(Bolton, MA) ; Mooney; David J.; (Sudbury, MA)
; Uzun; Oktay R.; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Family ID: |
1000006211410 |
Appl. No.: |
17/546489 |
Filed: |
December 9, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15321458 |
Dec 22, 2016 |
11229607 |
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PCT/US15/38601 |
Jun 30, 2015 |
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17546489 |
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62019284 |
Jun 30, 2014 |
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62082993 |
Nov 21, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/5052 20130101;
A61K 35/28 20130101; A61K 9/4816 20130101; C12N 11/10 20130101;
C12N 11/04 20130101; A61K 35/12 20130101; A61K 2035/124 20130101;
A61K 9/0019 20130101; A61K 9/5036 20130101 |
International
Class: |
A61K 9/48 20060101
A61K009/48; A61K 35/28 20060101 A61K035/28; A61K 9/00 20060101
A61K009/00; A61K 35/12 20060101 A61K035/12; C12N 11/10 20060101
C12N011/10; A61K 9/50 20060101 A61K009/50; C12N 11/04 20060101
C12N011/04 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole, or in part, by a
National Institutes of Health (NIH) R01 grant EB014703-03. The
Government has certain rights in the invention.
Claims
1-23. (canceled)
24. A method of administering at least one protein factor produced
by a cell to a subject in need thereof, the method comprising
administering to said subject a composition comprising a plurality
of hydrogel capsules, wherein at least 90% of said hydrogel
capsules in said composition comprise a cell and a hydrogel
encapsulating said cell, wherein said hydrogel encapsulating said
cell has a thickness of less than 20 microns.
25. The method of claim 24, wherein the cell is a mesencymal stem
cell (MSC) or a progenitor thereof.
26. The method of claim 24, wherein the hydrogel in each cell
containing hydrogel capsule is characterized by a stiffness of
about 0.1 to about 500 kPa.
27. The method of claim 24, wherein each cell containing hydrogel
capsule is characterized by a stiffness of about 10 kPa.
28. The method of claim 24, wherein said at least one protein
factor is naturally produced by said cell.
29. The method of claim 24, wherein said at least one protein
factor is not naturally produced by said cell, and wherein said
cell has been genetically engineered to produce said at least one
protein factor.
30. The method of claim 24, wherein said cell has been genetically
engineered to modify expression of at least one protein factor.
31. The method of claim 25, wherein said at least one protein
factor is a hematopoietic factor.
32. The method of claim 31, wherein said hematopoietic factor is
selected from the group consisting of stem cell factor (SCF),
interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6),
interleukin-7 (IL-7), granulocyte-macrophage colony stimulating
factor (GM-CSF), granulocyte colony stimulating factor (G-CSF),
macrophage colony stimulating factor (M-CSF), erythropoietin,
thrombopoietin, collagen-I, interleukin-11 (IL-11), angiopoietin-1
and transforming growth factor-beta (TGF-beta).
33. The method of claim 24, wherein the subject is a human.
34. The method of claim 25, wherein the number of hematopoietic
stem cells (HSCs) in the subject is increased.
35. The method of claim 25, wherein said at least one protein
factor is a cardiovascular regeneration factor.
36. The method of claim 35, wherein said cardiovascular
regeneration factor is selected from the group consisting of
vascular endothelial growth factor (VEGF), stromal cell derived
factor 1 (SDF-1), tumor necrosis factor-inducible gene 6 protein
(TSG-6), interleukin-6 (IL-6), interleukin-8 (IL-8), basic
fibroblast growth factor (bFGF or FGF-2), insulin-like growth
factor 1 (IGF-1), hepatocyte growth factor (HGF), thrombospondin-4,
secreted frizzled-related protein 2 (Sfrp2), matrix
metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinases
(TIMP) metallopeptidase inhibitor 2 (TIMP-2), monocyte chemotactic
protein 1 (MCP-1), thrombospondin 1 (TSP-1), chemokine (C-X-C
motif) ligand 6 (CXCL6) and interferon gamma-induced protein 10
(IP-10).
37. The method of claim 25, wherein said at least one protein
factor is a GVHD suppression factor.
38. The method of claim 37, wherein said GVHD suppression factor is
selected from the group consisting of transforming growth
factor-beta (TGF-beta), hepatocyte growth factor (HGF),
prostaglandin E2 (PGE2), galectin and indoleamine 2,3-dioxygenase
(IDO).
39. The method of claim 24, wherein the composition is administered
by a route selected from the group consisting of intravenous
infusion, intrabone infusion, intramuscular injection, subcutaneous
implantation, intraperitoneal injection, intracardial injection,
intratracheal administration, topical application and oral
administration.
40. The method of claim 39, wherein the composition is administered
by intravenous infusion.
41. The method of claim 34, wherein the subject has undergone bone
marrow transplantation or HSC transplantation.
42. The method of claim 34, wherein the subject suffers from
cancer, an immune deficiency disorder, or a blood disease.
43. The method of claim 42, wherein the cancer is a blood cancer or
a solid tumor cancer.
44-74. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/321,458, filed on Dec. 22, 2016; which is a 35 U.S.C. .sctn.
371 national stage filing of International Application No.
PCT/US2015/038601, filed on Jun. 30, 2015; which claims priority to
U.S. Provisional Application No. 62/019,284, filed on Jun. 30, 2014
and U.S. Provisional Application No. 62/082,993, filed on Nov. 21,
2014. The entire contents of each of the foregoing applications are
hereby incorporated herein by reference.
FIELD OF INVENTION
[0003] The present invention relates to hydrogel compositions for
cell delivery.
SEQUENCE LISTING
[0004] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 17, 2022, is named 117823-09504_SL.txt and is 3,105 bytes
in size.
BACKGROUND OF THE INVENTION
[0005] Delivering cells and their secretions to the body remains a
major challenge due to rapid clearance by physical and immune
barriers. There is a need for methods to shield exogenously
administered cells from the body's natural clearance mechanism.
Such methods would significantly improve the in vivo delivery of
biologics. While a bulk crosslinked hydrogel has been used for this
purpose, it has been difficult to inject the bulk gel into the
body, and the administration route has generally been limited to
subcutaneous.
[0006] Hematopoiesis is the formation and development of blood
cells. In embryos and fetuses, this process occurs in the liver,
spleen, thymus, lymph nodes, and bone marrow. After birth,
hematopoiesis occurs predominantly in the bone marrow. All blood
cell types, including erythrocytes and cells of the myeloid and
lymphoid lineages, are derived from multipotent hematopoietic stem
cells (HSCs). Various factors regulate HSC regeneration and
differentiation.
[0007] Bone marrow transplantation and HSC transplantation are used
to treat a number of diseases, e.g., hematological diseases,
immunodeficiencies, lysosomal storage disorders, and cancers. In
addition, blood transfusions are in demand for patients who have
undergone surgery, suffer from injuries, suffer from diseases such
as cancer, or suffer from injuries in sports arenas or
battlefields. There remains a need for methods of programming
hematopoiesis ex vivo and in vivo, e.g., to sustain in vivo
regeneration of blood or ex vivo production of blood. There is also
a need for methods of preserving long-term self-renewal of HSCs in
vivo, e.g., in diseased patients or patients who have undergone
transplantation.
SUMMARY OF THE INVENTION
[0008] The invention addresses these needs by providing
compositions that comprise capsules that comprise cross-linked
hydrogels that encapsulate individual cells, e.g., mesenchymal stem
cells (MSCs). The hydrogel encapsulated cells (also called hydrogel
capsules, micro-carriers, microgels or microparticles) may be
administered to a subject via a wide range of administration
routes, e.g., by intravenous infusion or injection. The hydrogel
encapsulated cells are also characterized by improved cell
viability and are capable of sustained secretion of protein
factors, such as factors that promote hematopoiesis, or factors
that promote cardiovascular regeneration in a subject.
[0009] The present invention is based, at least in part, on a
surprising discovery of a high-yield microfluidic technique to
encapsulate single cells in hydrogel capsules with a thin layer of
hydrogel. Specifically, it was discovered that contacting a cell
with a moiety comprising a cross-linking catalyst and allowing such
moiety to be adsorbed to the cell prior to hydrogel formation,
results in a high yield of cell encapsulation, i.e., produces a
composition with a high fraction of hydrogel capsules comprising a
cell. Such compositions also have a low fraction of empty hydrogel
capsules. This technique also allows for the production of
compositions with a high fraction of hydrogel capsules comprising a
single cell. Further, the thickness of the hydrogel layer in these
hydrogel capsules comprising a cell is small, e.g., 20 microns or
less. Accordingly, the mechanical properties and composition of the
hydrogel coating can be tuned at the single cell-level to control
biological functions of encapsulated cells in vitro and in vivo. A
composition comprising a high fraction of singly encapsulated cells
can be delivered in vivo via, for example, intravenous infusion.
Such compositions also show prolonged biodistribution in vivo and
secrete soluble protein factors that are useful for many
indications.
[0010] The invention is also based, at least in part, on the
discovery that release of protein factors from a hydrogel capsule
comprising a cell can be regulated by altering the mechanical
properties of the hydrogel layer. For example, it was discovered
that stiffness of the hydrogel layer encapsulating a cell, e.g., a
mesenchymal stem cell (MSC), regulates the release of protein
factors produced by the cell, e.g., hematopoietic factors that
regulate hematopoiesis and differentiation of hematopoietic stem
cells (HSCs). Specifically, it was shown that stiffness of the
hydrogel layer regulates exocytosis pathways in MSCs, e.g.,
exocytosis of factors, such as hematopoietic factors, from MSCs.
The hydrogel capsules of the present invention mechanically trigger
hematopoietic factor release from encapsulated cells, e.g., MSCs,
in vivo.
[0011] Accordingly, in one aspect, the present invention provides a
composition comprising a plurality of hydrogel capsules, wherein at
least 90%, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99%, of the hydrogel capsules in the composition comprise a cell
and a hydrogel encapsulating the cell, wherein the hydrogel
encapsulating the cell has a thickness of less than 20 microns,
e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2 or 1 microns.
[0012] In one embodiment, at least 70%, e.g., at least 75%, 80%,
85%, 90% or 95%, of the hydrogel capsules comprise a single
cell.
[0013] In another embodiment, the cell is a mesenchymal stem cell
(MSC) or a progenitor thereof, a hematopoietic stem cell (HSC) or a
progenitor thereof, or an endothelial progenitor cell.
[0014] In some embodiments, the hydrogel comprises at least one
polymer, e.g., a polymer selected from the group consisting of
alginate, agarose, poly(ethylene glycol dimethacrylate), polylactic
acid, polyglycolic acid, PLGA, gelatin, collagen, agarose, pectin,
poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone,
polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide),
poly(ethylene oxide), poly(allylamine), poly(acrylate),
poly(4-aminomethylstyrene), pluronic polyol, polyoxamer,
poly(uronic acid), poly(anhydride) and poly(vinylpyrrolidone). In a
specific embodiment, the polymer is alginate.
[0015] In a further embodiment, the polymer comprises polymer
chains cross-linked to each other using a divalent or trivalent
cation, e.g., Ca.sup.2+, Mg.sup.2+, Sr.sup.2+, Ba.sup.2+, Be.sup.2+
and Al.sup.2+. In a specific embodiment, the divalent cation is
Ca.sup.2+.
[0016] In certain embodiments, the diameter of the hydrogel capsule
comprising a single cell is between about 10 and 500 micron, e.g.,
between about 10 and about 200 micron, about 50 micron and about
300 micron, about 250 and about 500 micron, about 20 and about 80
micron, about 100 and about 400 micron, about 250 and about 450
micron and about 30 to about 150 micron. In a further embodiment,
the diameter of the hydrogel capsule comprising a single cell is
between about 25 and about 30 micron, e.g., about 25 and about 27
micron, about 26 and about 29 micron, about 27 and about 30
micron.
[0017] In one embodiment, the hydrogel comprises a first polymer
and a second polymer. In a specific embodiment, the first polymer
is alginate and the second polymer is collagen or fibrin.
[0018] In another aspect, the present invention also provides a
method of preparing a composition comprising a plurality of
hydrogel capsules. The method includes: a) contacting a cell with a
moiety capable of adhering to a cell and comprising a cross-linking
catalyst; and b) contacting the cell and the moiety with at least
one polymer comprising a plurality of polymer chains; wherein the
cross-linking catalyst catalyzes a reaction that cross-links the
plurality of polymer chains, thereby forming a composition
comprising a plurality of hydrogel capsules, wherein at least 90%
of the hydrogel capsules in the composition comprise a cell and a
hydrogel encapsulating the cell, wherein the hydrogel encapsulating
the cell has a thickness of less than 20 microns.
[0019] In one embodiment, the cross-linking catalyst is a divalent
or trivalent cation. In one embodiment, the divalent or trivalent
cation is selected from the group consisting of Ca.sup.2+,
Mg.sup.2+, Sr.sup.2+, Ba.sup.2+, Be.sup.2+ and Al.sup.2+. In a
further embodiment, the moiety is a nanonparticle, e.g., a
CaCO.sub.3 nanoparticle, a BaCO.sub.3 nanoparticle, or a SrCO.sub.3
nanoparticle.
[0020] In some embodiments, at least one polymer is selected from
the group consisting of alginate, agarose, poly(ethylene glycol
dimethacrylate), polylactic acid, polyglycolic acid, PLGA, gelatin,
collagen, agarose, pectin, poly(lysine), polyhydroxybutyrate,
poly-epsilon-caprolactone, polyphosphazines, poly(vinyl alcohol),
poly(alkylene oxide), poly(ethylene oxide), poly(allylamine),
poly(acrylate), poly(4-aminomethylstyrene), pluronic polyol,
polyoxamer, poly(uronic acid), poly(anhydride) and
poly(vinylpyrrolidone). In a further aspect, the at least one
polymer is alginate.
[0021] In some embodiments, the cell is a mesenchymal stem cell
(MSC) or a progenitor thereof, a hematopoietic stem cell (HSC) or a
progenitor thereof, or an endothelial progenitor cell. In a
specific embodiment, the mesenchymal stem cell (MSC) or a
progenitor thereof.
[0022] In a further aspect, the present invention also provides a
method of administering at least one protein factor produced by a
cell to a subject in need thereof. The method includes
administering to the subject a composition comprising a plurality
of hydrogel capsules, wherein at least 90%, e.g., at least 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of said hydrogel capsules
in the composition comprise a cell and a hydrogel encapsulating the
cell, wherein the hydrogel encapsulating the cell has a thickness
of less than 20 microns, e.g., less than 19, 18, 17, 16, 15, 14,
13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 microns.
[0023] In one embodiment, the cell is a mesencymal stem cell (MSC)
or a progenitor thereof.
[0024] In another embodiment, the hydrogel in each cell containing
hydrogel capsule is characterized by a stiffness of about 0.1 to
about 500 kPa, e.g., about 0.1 to about 10 kPa, about 0.5 to about
15 kPa, about 1 to about 15 kPa, about 5 to about 20 kPa, about 10
to about 50 kPa, about 20 to about 100 kPa, about 150 to about 300
kPa, about 100 to about 400 kPa, about 200 to about 450 kPa or
about 250 to about 500 kPa. In a further aspect, each cell
containing hydrogel capsule is characterized by a stiffness of
about 10 kPa, about 15 kPa, about 20 kPa, about 25 kPa, about 30
kPa, about 35 kPa, about 40 kPa, about 45 kPa, about 50 kPa, about
55 kPa, about 60 kPa, about 65 kPa, about 70 kPa, about 75 kPa,
about 80 kPa, about 85 kPa, about 90 kPa, about 95 kPa or about 100
kPa.
[0025] In one embodiment, at least one protein factor is naturally
produced by the cell. In another embodiment, the at least one
protein factor is not naturally produced by the cell and the cell
has been genetically engineered to produce the at least one protein
factor. In yet another embodiment, the cell has been genetically
engineered to modify expression of at least one protein factor. In
a further aspect, the at least one protein factor is a
hematopoietic factor.
[0026] In some embodiments, the hematopoietic factor is selected
from the group consisting of stem cell factor (SCF), interleukin-2
(IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7
(IL-7), granulocyte-macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF), macrophage colony
stimulating factor (M-CSF), erythropoietin, thrombopoietin,
collagen-I, interleukin-11 (IL-11), angiopoietin-1 and transforming
growth factor-beta (TGF-beta). In a specific aspect, the subject is
a human.
[0027] In one embodiment, the number of hematopoietic stem cells
(HSCs) in the subject is increased.
[0028] In some embodiments, the composition is administered by a
route selected from the group consisting of intravenous infusion,
intrabone infusion, intramuscular injection, subcutaneous
implantation, intraperitoneal injection, intracardial injection,
intratracheal administration, topical application and oral
administration. In a specific embodiment, the composition is
administered by intravenous infusion.
[0029] In some embodiments, the subject has undergone bone marrow
transplantation or HSC transplantation. In other embodiments, the
subject suffers from cancer, an immune deficiency disorder, or a
blood disease. In a further embodiment, the cancer is a blood
cancer or a solid tumor cancer. In another embodiment, the blood
cancer is leukemia, lymphoma or myeloma. In yet another embodiment,
the solid tumor cancer is selected from the group consisting of an
adrenocortical tumor, colorectal carcinoma, breast cancer, lung
cancer, ovarian cancer, uterine cancer, endometrial cancer,
cervical cancer, gliobastoma, colon cancer, stomach cancer,
pancreatic cancer, desmoid tumor, desmoplastic small round cell
tumor, endocrine tumor, Ewing sarcoma, hepatocellular carcinoma,
melanoma, neuroblastoma, osteosarcoma, retinoblastoma,
rhabdomyosarcoma, Wilms tumor, nasopharyngeal cancer, testicular
cancer, thyroid cancer, thymus cancer, gallbladder cancer, central
nervous system (CNS) cancer, bladder cancer and bile duct
cancer.
[0030] In another aspect, the blood disease is a disease selected
from the group consisting of thalassemia, aplastic anemia and
sickle cell anemia. In other embodiments, the immune deficiency
disorder is a disorder selected from the group consisting of
X-linked agammaglobulinemia (XLA), severe combined immunodeficiency
(SCID disorder), common variable immunodeficiency and
alymphocytosis.
[0031] In another aspect, the present invention provides a method
for treating or preventing a cardiovascular disease in a subject in
need thereof. The method includes administering to the subject a
composition comprising a plurality of hydrogel capsules, wherein at
least 90%, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99%, of said hydrogel capsules in the composition comprise a cell
and a hydrogel encapsulating the cell, wherein the hydrogel
encapsulating the cell has a thickness of less than 20 microns,
e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,
5, 4, 3, 2 or 1 microns.
[0032] In some embodiments, the cardiovascular disease is selected
from the group consisting of coronary artery disease,
cardiomyopathy, hypertensive heart disease, heart failure, cor
pulmonale, cardiac dysrhythmia, indocarditis, inflammatory
cardiomegaly, myocarditis, valvular heart disease, cerebrovascular
disease, peripheral arterial disease, congenital heart disease and
rheumatic heart disease.
[0033] In certain embodiments, the composition is administered by a
route selected from the group consisting of intravenous infusion,
intrabone infusion, intramuscular injection, subcutaneous
implantation, intraperitoneal injection, intracardial injection,
intratracheal administration, topical application and oral
administration. In a specific embodiment, the composition is
administered by intravenous infusion.
[0034] In another aspect, the present invention also provides a
method of increasing secretion of a protein factor by a cell. The
method includes a) contacting the cell with a moiety capable of
adhering to a cell and comprising a cross-linking catalyst; and b)
contacting the cell and the moiety with at least one polymer
comprising a plurality of polymer chains; wherein the cross-linking
catalyst catalyzes a reaction that cross-links said plurality of
polymer chains, thereby forming a composition comprising a
plurality of hydrogel capsules, wherein at least 90%, e.g., at
least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of the
hydrogel capsules in the composition comprise a cell and a hydrogel
encapsulating the cell, wherein the hydrogel encapsulating the cell
has a thickness of less than 20 microns, e.g., less than 19, 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
microns, and wherein the encapsulation of the cell causes an
increase in the secretion of a protein factor by the cell.
[0035] In some embodiments, the cell is a mesenchymal stem cell
(MSC).
[0036] In some embodiments, the protein factor is a hematopoietic
factor. In a further embodiment, the hematopoietic factor is
selected from the group consisting of stem cell factor (SCF),
interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6),
interleukin-7 (IL-7), granulocyte-macrophage colony stimulating
factor (GM-CSF), granulocyte colony stimulating factor (G-CSF),
macrophage colony stimulating factor (M-CSF), erythropoietin,
thrombopoietin, collagen-I, interleukin-11 (IL-11), angiopoietin-1,
and transforming growth factor-beta (TGF-beta).
[0037] In other embodiments, the hydrogel is characterized by a
stiffness of about 0.1 to about 500 kPa, e.g., about 0.1 to about
10 kPa, about 0.5 to about 15 kPa, about 1 to about 15 kPa, about 5
to about 20 kPa, about 10 to about 50 kPa, about 20 to about 100
kPa, about 150 to about 300 kPa, about 100 to about 400 kPa, about
200 to about 450 kPa or about 250 to about 500 kPa. In a specific
embodiment, the hydrogel is characterized by a stiffness of about
10 kPa, about 15 kPa, about 20 kPa, about 25 kPa, about 30 kPa,
about 35 kPa, about 40 kPa, about 45 kPa, about 50 kPa, about 55
kPa, about 60 kPa, about 65 kPa, about 70 kPa, about 75 kPa, about
80 kPa, about 85 kPa, about 90 kPa, about 95 kPa or about 100
kPa.
[0038] In some embodiments, the amount of the hematopoietic factor
secreted by the cell is at least 1.5-fold greater than the amount
of hematopoietic factor secreted by the cell prior to step a.
[0039] In a further aspect, the present invention provides a
composition comprising a plurality of hydrogel capsules, wherein at
least 70%, e.g., at least 75%, 80%, 85%, 90% or 95%, of the
hydrogel capsules comprise a single cell and a hydrogel
encapsulating the cell, wherein the hydrogel encapsulating the cell
has a thickness of less than 20 microns, e.g., less than 19, 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
microns.,
[0040] In some embodiments, at least 90%, e.g., at least 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98% or 99%, of the hydrogel capsules in
the composition comprise a cell and a hydrogel encapsulating the
cell.
[0041] In some embodiments, the cell is a mesenchymal stem cell
(MSC) or a progenitor thereof, a hematopoietic stem cell (HSC) or a
progenitor thereof, or an endothelial progenitor cell. In a
specific embodiment, the cell is a mesenchymal stem cell (MSC) or a
progenitor thereof.
[0042] In certain embodiments, the hydrogel comprises at least one
polymer, e.g., the polymer selected from the group consisting of
alginate, agarose, poly(ethylene glycol dimethacrylate), polylactic
acid, polyglycolic acid, PLGA, gelatin, collagen, agarose, pectin,
poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone,
polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide),
poly(ethylene oxide), poly(allylamine), poly(acrylate),
poly(4-aminomethylstyrene), pluronic polyol, polyoxamer,
poly(uronic acid), poly(anhydride) and poly(vinylpyrrolidone). In a
further embodiment, the polymer is alginate.
[0043] In some embodiments, the polymer comprises polymer chains
cross-linked to each other using a divalent or trivalent cation.
For example, the divalent or trivalent cation is selected from the
group consisting of Ca.sup.2+, Mg.sup.2+, Sr.sup.2+, Ba.sup.2+,
Be.sup.2+ and Al.sup.2+. In a specific embodiment, the divalent
cation is Ca.sup.2+.
[0044] In certain embodiments, the diameter of the hydrogel capsule
comprising a single cell is between about 10 and 500 micron, e.g.,
10-100, 100-200, 200-300 or 400-500 micron. In a specific
embodiment, the diameter of the hydrogel capsule comprising a
single cell is between about 25 and about 30 micron, about 25 and
about 40 micron, about 25 and about 50 micron or about 30 and about
60 micron.
[0045] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a set of schematics showing the regulation of
secretion of hematopoietic factors from MSCs by matrix stiffness as
well as single MSC encapsulation into a hydrogel and subsequent
infusion of the hydrogel to a subject to program hematopoiesis in
vivo.
[0047] FIG. 2A is a set of schematics showing the structure of
alginate hydrogels ionically crosslinked with Ca.sup.2+ (see, e.g.,
Sun et al. 2012, Nature 489, 133-136).
[0048] FIG. 2B is a graph showing gelation kinetics of 1% alginate
with different Ca.sup.2+ concentrations and molecular weights. The
rheological measurements of alginate hydrogels during gel formation
were performed at 37.degree. C. with a frequency of 0.05 Hz and a
stress of 1 Pa.
[0049] FIG. 3A is a graph showing the effect of matrix stiffness on
CD34+ subpopulations in vitro. Matrix stiffness decreases the
number of HSC-enriched subpopulations. Maximum=331, IC.sub.50=1384
Pa, Hill coefficient=-0.5.
[0050] FIG. 3B is a bar graph showing that matrix stiffness
regulates the release of soluble factors from MSCs that impact
HSC/P differentiation in vitro. MSC:CD34+ ratio=5:1. *P<0.05
from one-way ANOVA with Tukey's HSD test. The absoluate cell number
was normalized by the total 10,000 cell input. n.gtoreq.3.
[0051] FIG. 4A is an image showing encapsulation of single MSCs in
alginate gel droplets. Green=FITC-alginate (10/60), Red=Phalloidin
(F-actin), Blue=DAPI (nucleus).
[0052] FIG. 4B is an image, schematic and set of equations showing
an AFM measurement scheme. (Top) Droplets (green) seeded in a
micro-well made of PDMS. (Bottom) AFM indentation schematics and
calculation of Young's Modulus (E) with the Hertz model.
[0053] FIG. 4C is a bar graph showing the Young's Modulus (E) of
gel droplets (20 um diameter) encapsulated with live single MSCs
with different initial Ca.sup.2+ concentrations. n>5
droplets.
[0054] FIG. 5 is a schematic showing the methods used to
characterize and quantify the hematopoietic factors released from
MSCs in different matrix rigidities, as well as the methods used to
delineate the mechanisms behind the stiffness-dependent factor
release.
[0055] FIG. 6 is a schematic showing the encapsulation of single
cells, e.g., MSCs, in alginate droplets with substrates of
different stiffness, followed by infusion into a subject to program
hematopoiesis in vivo.
[0056] FIG. 7 is a set of images and graphs showing the
characterization of single hMSCs encapsulated in alginate
beads.
[0057] FIG. 8 is a set of images and graphs showing optimization
for in vivo delivery alginate beads with stiffness are resistant
under in vitro shear.
[0058] FIG. 9 is a set of images and graphs showing live imaging of
alginate beads in C57BL6/J mice.
[0059] FIG. 10A is a set of images showing live imaging of mouse
MSC-luciferase in C57BL6/J mice.
[0060] FIG. 10B is a set of images and graphs showing prolonged
biodistribution of cells by encapsulation.
[0061] FIG. 11 is a set of images showing that prolonged
biodistribution requires cells to be encapsulated.
[0062] FIG. 12 is an image showing that injection of the beads into
the left ventricle of the heart led to localization in the
liver.
[0063] FIG. 13 is a set of graphs showing live imaging of human
MSC-luciferase in NSG mice.
[0064] FIG. 14 is a set of schematics and graphs showing that soft
substrate stiffness maximized HSC-enriched subpopulation, e.g., in
gels comprising HSCs.
[0065] FIG. 15 is a set of graphs and images showing the effect of
stiffness of alginate gel encapsulating hMSCs on the HSC-enriched
number due to protein secretion.
[0066] FIG. 16A is a part of a schematic of a cell encapsulation
procedure, showing cells coated with calcium carbonate
nanoparticles.
[0067] FIG. 16B is a a part of a schematic of a cell encapsulation
procedure, showing that excess nanoparticles are removed, and
nanoparticle-coated cells are mixed with alginate.
[0068] FIG. 16C is a a part of a schematic of a cell encapsulation
procedure, showing that the suspension and a fluorinated
oil-surfactant-acetic acid solution are injected into a
microfluidic device. An aqueous-in-oil emulsion is formed at the
T-junction in the device. Acetic acid diffuses into the aqueous
phase. Only cell- and nanoparticle-containing droplets
cross-link.
[0069] FIG. 16D is a part of a schematic of a cell encapsulation
procedure, showing that the emulsion is mixed with cell medium and
broken by addition of perfluoro-octanol.
[0070] FIG. 17A is an image showing D1 cells directly encapsulated
in alginate without pre-coating with nanoparticles and stained for
viability (gray circles, alginate; bright circles, live cells).
[0071] FIG. 17B is an image showing D1 cells pre-coated with
nanoparticles and then encapsulated in alginate (gray circles,
alginate; bright circles, live cells).
[0072] FIG. 17C is a confocal slice of encapsulated D1 cell. Outer
circle, alginate; middle circle, actin; inner circle, nuclei.
[0073] FIG. 17D is a graph showing the thickness of hydrogel layer,
measured at multiple locations around cells, for 9 encapsulated D1
cells.
[0074] FIG. 17E is a histogram of alginate intensity per pixel
taken from confocal images of 16 different cell-encapsulating
alginate capsules, fabricated using the pre-coating method. The
single peak indicates homogeneity within the capsules.
[0075] FIG. 17F is a histogram of alginate intensity from 40,475
events, consisting of the encapsulation output after pre-coating
cells with nanoparticles.
[0076] FIG. 17G is a histogram of initial number of cell(s) per
alginate microgel for three different cell types encapsulated after
pre-coating with nanoparticles.
[0077] FIG. 17H is a graph showing the fraction of D1 cells, OP9
cells, and OECs encapsulated in microgels (efficiency) by direct
encapsulation, direct encapsulation followed by FACS to generate a
pure population, and pre-coating with CaCO.sub.3 nanoparticles
before encapsulation.
[0078] FIG. 17I is a graph showing the fraction of alginate beads
containing encapsulated D1 and OP9 cells and OECs (yield) by direct
encapsulation, direct encapsulation followed by FACS, and
pre-coating with CaCO.sub.3 nanoparticles.
[0079] FIG. 17J is a graph showing viability of encapsulated cells
1 day and 3 days after encapsulation using precoating with
nanoparticles (for D1s, OP9s, and OECs), with direct injection
without pre-coating followed by a FACS sort (for D1s), and in bulk
hydrogels (for OECs).
[0080] FIG. 18A is set of fluorescent and matching bright field
images of D1s encapsulated in 54 kDa alginate.
[0081] FIG. 18B is a set of fluorescent and matching bright field
images of D1s encapsulated in 139 kDa alginate.
[0082] FIG. 18C is a set of fluorescent and matching bright field
images of D1s encapsulated in 232 kDa alginate. The alginate is
labeled with a red fluorophore in FIGS. 18A and B, and a green
fluorophore in FIG. 18C.
[0083] FIG. 18D is a graph showing the comparison of the fraction
of cells encapsulated (efficiency) as a function of polymer
molecular weight.
[0084] FIG. 18E is a graph showing the fraction of microgels
containing cells (yield) as a function of polymer molecular
weight.
[0085] FIG. 18F is a graph showing the fraction of viable cells
over time as a function of polymer molecular weight.
[0086] FIG. 18G is a graph showing the hydrogel capsule size as a
function of polymer molecular weight. Differences in the fraction
of cells encapsulated and the fraction of viable cells depending on
molecular weight were not significantly different (chi square
test). Initial hydrogel capsule size difference was significant
(1-way ANOVA, p<0.05). Yield in 232 kDa alginate was
significantly lower than other conditions, as analyzed by chi
square test, p<0.01.
[0087] FIG. 18H is a graph showing the average number of cells per
microgel.
[0088] FIG. 18I is a graph showing the microgel size after 3 days
of culture (1-way ANOVA, p<0.01) are shown.
[0089] FIG. 18J is a fluorescent image (left) and a brightfield
image (right) of a cell leaving its microgel. Red, alginate; blue,
nucleus; green, actin; scale bar: 50 microns.
[0090] FIG. 18K is a graph showing the fraction of cells that
egressed from microgels formed from alginate of different molecular
weights into surrounding collagen gel. Differences between
conditions were statistically significant (chi-square test, df=2,
p<0.01).
[0091] FIG. 18L is a graph showing the elastic moduli of microgels
that were treated post-encapsulation with solutions containing
different concentrations of calcium chloride.
[0092] FIG. 19A are confocal images of alginate-collagen microgels,
scale bar 20 microns.
[0093] FIG. 19B are confocal images of alginate-fibrin microgels,
scale bar 20 microns.
[0094] FIG. 19C is a histogram of alginate fluorescence per pixel
in collagen-alginate microgels from confocal slices from 7
different microgels.
[0095] FIG. 19D is a scatter plot of polymer concentration per
collagen-alginate microgel, as assessed by mean fluorescent
intensity, from 168 microgels.
[0096] FIG. 19E is a histogram of alginate fluorescence per pixel
in fibrin-alginate microgels from confocal slices from 29 different
microgels.
[0097] FIG. 19F is a histogram of alignate fluorescence per 10
pixels in fibrin-alginate microgels from confocal slices from 29
different microgels.
[0098] FIG. 19G is a scatter plot of polymer concentration per
fibrin-alginate microgel, as assessed by mean fluorescent
intensity, from 103 microgels.
[0099] FIG. 19H is a fluorescent image of fibrin-encapsulated D1
cell.
[0100] FIG. 19I is a transmitted light image of the cell in FIG.
19H. Scale bar 50 microns.
[0101] FIG. 19J is a histogram of fibrin concentration per fibrin
microgel from 44 different microgels.
[0102] FIG. 19K is a graph showing encapsulation efficiency and
yield in collagen-alginate, fibrin-alginate, and fibrin
microgels.
[0103] FIG. 19L is a graph showing viable fraction of encapsulated
D1s in hydrogel capsules of different polymer compositions after 24
hours and 72 hours of culture. ** denotes p<0.01. These figures
show encapsulation of D1s in hybrid hydrogel capsules/various
polymers.
[0104] FIG. 20A is a schematic of a culture apparatus, showing
alginate-encapsulated cells settled in PDMS microwells of different
diameters.
[0105] FIG. 20B is a graph showing the number of hydrogel capsules
per 100-um deep microwells as a function of microwell area.
[0106] FIG. 20C is a graph of hydrogel capsule area density as a
function of microwell area, in 100-.mu.m deep (circles) and
200-.mu.m deep (triangles) microwells. *=p<0.05.
[0107] FIG. 20D is a set of images showing cells encapsulated in
FITC-tagged alginate, stained after 6 days of culture, with actin
and an ALP marker (white); scale bar=100 .mu.m.
[0108] FIG. 20E is a set of confocal images showing some cells
still inside hydrogel capsules while others have egressed and
proliferated; scale bar=20 .mu.m.
[0109] FIG. 20F is a log-log plot of alkaline phosphatase
expression by encapsulated D1 cells after 6 days of culture as a
function of alginate fluorescence, per microwell, r=0.76,
p<0.01. Black line shows least-square fit. The symbols in FIG.
20F indicate different microwell sizes that correspond to those in
FIG. 20B. These figures show the seeding and differentiation of
encapsulated D1s in a PDMS microwell system.
[0110] FIG. 21A is a graph showing calcium adsorption to cells as a
function of concentration of calcium carbonate nanoparticles in
suspension incubated with cells.
[0111] FIG. 21B is a schematic of a microfluidic device used for
encapsulations.
[0112] FIG. 21C is a graph showing the concentration of calcium
released from calcium carbonates nanoparticles in a 3.0 mg/mL
suspension upon exposure to cell medium, acetic acid, alginate, and
a combination of acetic acid and alginate.
[0113] FIG. 21D is a graph showing cell viability as a function of
duration of exposure to hydrofluoroether 7500 with 1%
fluorosurfactant, as assessed by Alamar Blue and normalized to the
highest AFU value.
[0114] FIG. 21E is a graph showing relative intracellular calcium
levels in non-encapsulated cells (cells), encapsulated cells, and
cells directly exposed to CaCO.sub.3 nanoparticles (cells with
CaCO.sub.3). For this assay, cells were incubated in their
respective conditions for 24 hours. *=p<0.05, **=p<0.01.
[0115] FIG. 21F are representative brightfield and fluorescent
images of OECs encapsulated in rhodamine-labeled alginate.
[0116] FIG. 21G are representative brightfield and fluorescent
images of OP9 cells encapsulated in rhodamine-labeled alginate.
Scale bar=50 um.
[0117] FIG. 22A is a set of images of D1 cells encapsulated in 54
kDa alginate after 3 days of culture.
[0118] FIG. 22B is a set of images of D1 cells encapsulated in 139
kDa alginate after 3 days of culture.
[0119] FIG. 22C is a set of images of D1 cells encapsulated in 232
kDa alginate after 3 days of culture. Scale bar, 30 microns. Top:
fluorescence; bottom: brightfield.
[0120] FIG. 22D is a graph showing measurements of bulk modulus, as
assessed by an Instron mechanical apparatus, as a function of
nano-scale modulus of the same hydrogel, as assessed by AFM.
[0121] FIG. 23A is a schematic of microfluidic device used for
fabrication of fibrin and fibrin-alginate hydrogel capsules.
[0122] FIG. 23B is a representative image of a collagen-alginate
hydrogel capsule following EDTA treatment.
[0123] FIG. 23C is a representative image of a fibrin-alginate
hydrogel capsule following EDTA treatment.
[0124] FIG. 24A is a set of images showing the effects of in vitro
shear force on the microscale integrity of soft hydrogel capsules
(E .about.300 Pa).
[0125] FIG. 24B is a diagram showing the results of flow cytometry
analysis of human MSCs encapsulated in 139-kDa alginate hydrogel
capsule revealed .about.70% efficiency and .about.75% yield.
[0126] FIG. 24C is a bar graph showing the biodistribution of human
MSCs overexpressing Firefly luciferase with or without hydrogel
encapsulation after 24 hours of intravenous injection. The
bioluminescence signals from lungs were measured using IVIS at 24
hours post-injection and normalized against those at 30 min.
[0127] FIG. 24D is a bar graph showing IL-6 secretion of singly
encapsulated human MSCs in vitro after 1 day of culture.
[0128] FIG. 24E is a bar graph showing levels of IL-6 secreted into
blood plasma by human MSCs encapsulated into 139-kDa alginate 4
hours after injection.
[0129] FIG. 25A is a set of representative bioluminescence images
showing the biodistribution of mMSCs overexpressing Firefly
Luciferase with or without hydrogel encapsulation after intravenous
injection. "C in M": Encapsulated cells ("Cell in Microgel"),
"C+M": Cells mixed with empty hydrogel capsules ("Cell+
Microgel").
[0130] FIG. 25B is a graph showing the clearance kinetics of singly
encapsulated mMSCs after intravenous injection compared to cells
alone, cells mixed with hydrogel capsules, and empty hydrogel
capsules. For each group, all the values were normalized by the
value at 30 min. The signals from lungs were measured. The
fluorescence signals were measured for empty hydrogel capsules,
while the luminescence signals were measured for hydrogel capsules
that contained cells. The data were fit to
Y=(1.00-Plateau)*exp(-k*t)+Plateau. For each group, t.sub.1/2
(ln(2)/k, hr) is: Empty Microgel=142.6; Cell in Mlcrogel=30.74;
Cell=2.59; Cell+Empty Microgel=2.85, n>5 recipients.
[0131] FIG. 25C is a graph showing blood plasma levels of Gaussia
Luciferase produced over 300 hours as indicated by area under
curve. Student's T-test, *P<0.05, n>6 recipients.
[0132] FIG. 25D is a bar graph showing blood plasma levels of human
IL-6 after intravenous injection of hMSCs into
NOD/SCID/IL2.gamma..sup.-/- (NGS) mice. Total human IL-6 level in
blood (pg/ml) over 24 hours after injection of 1 million hMSCs per
mouse. Student's T-test, **P<0.005, n>4 recipients.
[0133] FIG. 26A is a graph showing dose response of IL-6 secretion
induced by exogenous INF-.gamma. (100 ng/mL) in human primary bone
marrow MSCs encapsulated in alginate hydrogels of different
stiffness.
[0134] FIG. 26B is a graph showing the kinetics of MCP-1 (CCL2)
secretion in the presence or absence of TNF-.alpha. from human
primary bone marrow MSCs encapsulated in alginate hydrogels of
different stiffness.
[0135] FIG. 26C is a set of two graphs showing the kinetics of
secretion of hematopoietic factors from human primary bone marrow
MSCs encapsulated in alginate hydrogels of different stiffness.
Left panel is a graph showing the kinetics of secretion of SCF
following treatment with LPS (20,000 ng/mmL). Right panel is a
graph showing the kinetics of secretion of TGF-.beta. from
untreated cells.
[0136] FIG. 27A is a schematic showing the structure of a DNA
plasmid used to transduce mouse D1 MSCs. The DNA plasmid contains a
secreted form of Gaussia Luciferase and CFP. Both genes are
separated by IRES and their expression is driven by the
constitutive promoter SV40.
[0137] FIG. 27B is a graph showing the total amount of Gaussia
Luciferase secreted from the mouse D1 MSCs transduced by the DNA
plasmid shown in FIG. 27A and encapsulated in alginate hydrogels of
different stiffness.
[0138] FIG. 27C is a bar graph showing that secretion of Gaussia
Luciferase from the transduced mouse D1 MSCs is partially
suppressed by treatment with myosin-II inhibitor blebbistatin.
DETAILED DESCRIPTION OF THE INVENTION
[0139] The present invention provides hydrogel capsules containing
cells, e.g., single cells, and methods of preparing as well as
using them.
[0140] The hydrogel capsules and methods described herein provide
certain advantages over existing hydrogels and methods of cell
delivery. The hydrogel capsules of the invention provide a cell
delivery vehicle that is infusible/injectable (like a cell
suspension), but that, unlike a cell suspension, permits the
control of cells via matrix/gel mechanics. For example, by
encapsulating and surrounding one single cell or a few cells in a
matrix (e.g., comprising certain mechanical properties, such as
stiffness), the cell(s) is surrounded on all sides by the matrix
that is instructive to the cell(s) and that leads to certain
biological behaviors. In other examples, with the cell(s)
surrounded on all sides by the matrix, the matrix is obstructive to
the cell(s), e.g., and prevents cell-cell contact, cell
growth/proliferation, and/or contact with endogenous cells (e.g.,
immune cells) from the body.
[0141] The hydrogel capsules of the invention are capable of
shielding cells, e.g., exogenously administered cells (e.g.,
encapsulated in the micro-carriers), from clearance by the body,
e.g., by immune clearance or metabolism. For example, the hydrogel
matrix masks a human leukocyte antigen (HLA) molecule on the
encapsulated cell such that the encapsulated cell is capable of
evading the host immune system. The encapsulated cells, e.g.,
secrete factors, e.g., hematopoietic factors or factors that aid in
recovery of damaged tissue (e.g., heart tissue). Thus, the hydrogel
micro-carriers provide sustained/extended release of such
clinically relevant factors.
[0142] Further, the hydrogel micro-carriers are
injectable/infusible and do not require more invasive methods, such
as implantation. For example, the hydrogels are small enough to be
intravenously administered/infused, unlike previously described
gels, which have a larger size that would block blood vessels if
infused.
I. Hydrogel Capsules Containing Cells
[0143] In certain embodiments, the present invention provides a
composition comprising a plurality of hydrogel capsules, wherein at
least 90% of the hydrogel capsules in the composition comprise a
cell and a hydrogel encapsulating the cell, wherein the hydrogel
encapsulating the cell has thickness of less than 20 microns. For
example, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
99.5% of the hydrogel capsules in the composition may comprise a
cell, e.g., one or more cells, such as 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 25, 30 or more cells.
[0144] In other embodiments, the present invention also provides a
composition comprising a plurality of hydrogel capsules, wherein at
least 70% of the hydrogel capsules comprise a single cell and a
hydrogel encapsulating the cell, wherein the hydrogel encapsulating
the cell has a thickness of less than 20 microns. For example, at
least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 99.5% of the hydrogel capsules may comprise a
single cell.
[0145] In some embodiments, the hydrogel encapsulating the cell has
thickness of less than 20 microns, e.g., less than 19, 18, 17, 16,
15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 microns.
[0146] The hydrogels described herein comprise a polymer, e.g., an
alginate, agarose, poly(ethylene glycol dimethacrylate), polylactic
acid, polyglycolic acid, PLGA, gelatin, pectin, collagen, agarose,
poly(lysine), polyhydroxybutyrate, poly-epsilon-caprolactone,
polyphosphazines, poly(vinyl alcohol), poly(alkylene oxide),
poly(ethylene oxide), poly(allylamine), poly(acrylate),
poly(4-aminomethylstyrene), pluronic polyol, polyoxamer,
poly(uronic acid), poly(anhydride) or poly(vinylpyrrolidone). In
one embodiment, the hydrogel comprises an alginate.
[0147] Alginates are versatile polysaccharide based polymers that
may be formulated for specific applications by controlling the
molecular weight, rate of degradation and method of hydrogel
formation. Coupling reactions can be used to covalently attach
bioactive epitopes, such as the cell adhesion sequence RGD to the
polymer backbone. Alginate polymers are formed into a variety of
scaffold types. Injectable/infusable hydrogels can be formed from
low MW alginate solutions upon addition of a cross-linking agents,
such as calcium ions, while macroporous hydrogels are formed by
lyophilization of high MW alginate discs. Differences in hydrogel
formulation control the kinetics of hydrogel degradation. Release
rates of morphogens or other bioactive substances from alginate
hydrogels is controlled by the formulation to present morphogens in
a spatially and temporally controlled manner. This controlled
release not only eliminates systemic side effects and the need for
multiple injections/infusions, but can be used to create a
microenvironment that activates host cells at a hydrogel implant
site and transplanted cells seeded onto/into a hydrogel.
##STR00001##
[0148] The hydrogel comprises a biocompatible polymer matrix that
is optionally biodegradable in whole or in part. Examples of
materials which can form hydrogels include polylactic acid,
polyglycolic acid, PLGA polymers, alginates and alginate
derivatives, gelatin, collagen, agarose, pectin, natural and
synthetic polysaccharides, polyamino acids such as polypeptides
particularly poly(lysine), polyesters such as polyhydroxybutyrate
and poly-epsilon.-caprolactone, polyanhydrides; polyphosphazines,
poly(vinyl alcohols), poly(alkylene oxides) particularly
poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates),
modified styrene polymers such as poly(4-aminomethylstyrene),
pluronic polyols, polyoxamers, poly(uronic acids),
poly(vinylpyrrolidone) and copolymers of the above, including graft
copolymers.
[0149] The hydrogels may be fabricated from a variety of synthetic
polymers and naturally-occurring polymers such as, but not limited
to, collagen, fibrin, hyaluronic acid, agarose, pectin, and
laminin-rich gels. One preferred material for the hydrogel is
alginate or modified alginate material. Alginate molecules are
comprised of (1-4)-linked .beta.-D-mannuronic acid (M units) and a
L-guluronic acid (G units) monomers, which can vary in proportion
and sequential distribution along the polymer chain. Alginate
polysaccharides are polyelectrolyte systems which have a strong
affinity for divalent cations (e.g., Ca.sup.+2, Mg.sup.+2,
Ba.sup.+2) or trivalent cations (e.g., Al.sup.3+) and form stable
hydrogels when exposed to these molecules. See Martinsen A., et
al., Biotech. & Bioeng., 33 (1989) 79-89.) For example, calcium
cross-linked alginate hydrogels are useful as a matrix for cells,
such as MSCs.
[0150] An exemplary hydrogel utilizes an alginate or other
polysaccharide of a relatively low molecular weight, preferably of
size which, after dissolution, is at the renal threshold for
clearance by humans, e.g., the alginate or polysaccharide is
reduced to a molecular weight of 1000 to 80,000 daltons.
Preferably, the molecular mass is 1000 to 60,000 daltons,
particularly preferably 1000 to 50,000 daltons. It is also useful
to use an alginate material of high guluronate content since the
guluronate units, as opposed to the mannuronate units, provide
sites for ionic crosslinking through divalent cations to gel the
polymer. U.S. Pat. No. 6,642,363, incorporated herein by reference,
discloses methods for making and using polymers containing
polysachharides such as alginates or modified alginates that are
particularly useful for cell transplantation and tissue engineering
applications.
[0151] In some examples, the polymer of the hydrogel is modified
with (e.g., covalently or noncovalently with) a cell adhesive
peptide. Exemplary cell adhesive peptides include
arginine-glycine-aspartate (RGD), RGDS (SEQ ID NO: 1), LDV, REDV
(SEQ ID NO: 2), RGDV (SEQ ID NO: 3), LRGDN (SEQ ID NO: 4), IKVAV
(SEQ ID NO: 5), YIGSR (SEQ ID NO: 6), PDSGR (SEQ ID NO: 7),
RNIAEIIKDA (SEQ ID NO: 8), RGDT (SEQ ID NO: 9), DGEA (SEQ ID NO:
10), and VTXG (SEQ ID NO: 11). In some examples, the cell adhesive
peptide comprises the RGD amino acid sequence.
[0152] The hydrogels of the invention may be porous or non-porous.
For example, the hydrogels may be nanoporous having a diameter of
less than about 10 nm; microporous wherein the diameter of the
pores are preferably in the range of about 100 nm-20 .mu.m; or
macroporous wherein the diameter of the pores are greater than
about 20 .mu.m, more preferably greater than about 100 .mu.m and
even more preferably greater than about 400 .mu.m. Some methods of
preparing hydrogels, e.g., hydrogel micro-carriers, are described
herein. Other methods of preparing porous hydrogel products are
known in the art. (See, e.g., U.S. Pat. No. 6,511,650, incorporated
herein by reference).
[0153] A hydrogel capsule in the compostions of the invention may
comprise 1-50, e.g., 1-40, 1-30, 1-20, 1-10, 9, 8, 7, 6, 5, 4, 3,
2, 1 cells (e.g., mesenchymal stem cells (MSCs) or progenitors
thereof, HSCs or progenitor thereof, or endothelial progenitor
cells.
[0154] Hydrogel encapsulation of a cell prolongs the life/viability
of a cell by protecting the cell from immune system mediated cell
death, e.g., transplant rejection mechanisms, as well as instructs
cells regarding function, e.g., secretion of paracrine factors. For
example, hydrogels with specified matrix stiffnesses regulate
exocytosis of paracrine factors from cells such as mesenchymal stem
cells (MSCs). Mesenchymal stem cells (MSCs) are multipotent stromal
cells that can differentiate into a number of different cell types,
including osteoblasts, adipocytes, and chondrocytes. In some
embodiments, MSCs comprise multipotent cells derived from
non-marrow tissues, e.g., umbilical cord blood, adipose tissue,
adult muscle, corneal stroma, human peripheral blood, amniotic
fluid, or dental pulp of deciduous baby teeth.
[0155] In some embodiments, the hydrogel in the compositions of the
invention is characterized by a stiffness of 1 Pa-1000 kPa, e.g.,
1-10,000 Pa, 10-1000 Pa, 10-500 Pa, 10-100 Pa, 0.1-500 kPa, 1-1000
kPa, 1-500 kPa, 5-1000 kPa, 5-500 kPa, 0.1-500 kPa, 0.1-100 kPa,
1-500 kPa, 1-100 kPa, 5-500 kPa, 5-100 kPa, or 1-50 kPa, e.g.,
about 1, 5, 10, 15, or 20 kPa.
[0156] The polymer in the hydrogel capsule is crosslinked via
covalent or noncovalent cross-links. For example, the polymer is
crosslinked ionically (i.e., non-covalently) with a divalent or
trivalent cation, such as Ca.sup.2+, Mg.sup.2+, Sr.sup.2+,
Ba.sup.2+, Be.sup.2+ and Al.sup.3+.
[0157] In some examples, the stiffness of the hydrogel
encapsulating a cell, e.g., an MSC, induces secretion of a protein
factor, e.g., a hematopoietic factor, from the cell, e.g., an MSC.
Exemplary protein factors secreted by the cell may include
hematopoietic factors, cardiovascular regeneration factors, and
graft versus host disease (GVHD) suppression factors. Exemplary
hematopoietic factors, cardiovascular regeneration factors, and
graft versus host disease (GVHD) suppression factors are described
herein. Exemplary hematopoietic factors comprise a stem cell factor
(SCF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-6
(IL-6), interleukin-7 (IL-7), granulocyte-macrophage colony
stimulating factor (GM-CSF), granulocyte colony stimulating factor
(G-CSF), macrophage colony stimulating factor (M-CSF),
erythropoietin, thrombopoietin, collagen-I, interleukin-11 (IL-11),
angiopoietin-1, or transforming growth factor-beta (TGF-beta). The
hydrogel may increase the amount of a protein factor, e.g., a
hematopoietic factor, secreted by the cell, e.g., an MSC, by at
least 1.5-fold (e.g., at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 30, 40, 50, 100-fold, or greater) compared to that of the MSC
prior to exposure to the hydrogel.
[0158] A cell encapsulated by a hydrogel, e.g., an MSC, may secrete
at least one protein factor, e.g., a hematopoietic factor, that is
naturally produced by the cell. The cell may also be genetically
engineered to modify or eliminate the expression of one or more
protein factor that the cell may naturally secrete. For example,
the expression of protein factors by such cells may be modified by
using techniques known to one of skill in the art, such as plasmid
overexpression, RNA interference or CRISPR/Cas9 genome editing.
[0159] Alternatively, a cell may secrete at least one protein
factor that is not naturally produced by the cell. In such cases,
the cell, e.g., an MSC, may be genetically engineered to produce
and secrete any protein factor, e.g., using plasmid
overexpression.
[0160] In some embodiments, a cell encapsulated in the hydrogel
capsules of the invention, e.g., an MSC, is capable of
immunomodulation. For example, the encapsulated cell, e.g., an MSC,
may secrete immunomodulatory factors, e.g., indoleamine
2,3-dioxygenase (IDO), prostaglandin E(2) (PGE(2)), nitric oxide
(NO), histocompatibility leucocyte antigen-G (HLA-G), transforming
growth factor (TGF)-.beta., interferon (IFN)-.gamma. and
interleukin (IL)-1.beta.. As the immunomodulatory properties of a
cell, e.g., an MSC, may depend on its priming with inflammatory
factors, a cell may be exposed to priming factors, such as
TNF-.alpha. and IFN-.alpha., prior to encapsulation. Alternatively,
the priming factors, such as TNF-.alpha. and IFN-.alpha., may be
included in the hydrogel, e.g., an alginate hydrogel, encapsulating
a cell, e.g., an MSC. Alternatively, toll like receptor ligands may
be covalently coupled to the polymer, e.g., an alginate polymer,
used for encapsulation, in order to provide constant stimulation to
the encapsulated cells.
[0161] In another embodiment, a cell comprised in the hydrogel
capsules of the invention may be an MSC-derived induced pluripotent
stem cell (an iPS cell).
[0162] In some embodiments, an MSC comprised in the hydrogel
capsules of the present invention is a human MSC. In some examples,
a cell (e.g., MSC, HSCs/progenitor, or endothelial progenitor) is
autologous or heterologous, e.g., allogeneic. For example, a cell
(e.g., MSC, HSCs/progenitor, or endothelial progenitor) is
autologous. In another example, a cell (e.g., MSC, HSCs/progenitor,
or endothelial progenitor) is allogeneic, e.g., a related
allogeneic cell or an unrelated allogeneic cell. For example, the
hydrogel micro-carrier provides a physical barrier between the
encapsulated cell and the body's immune cells, thereby
reducing/preventing an adverse host immune response that, in other
cases (e.g., where an exogenous cell is administered
non-encapsulated) would lead to exogenous cell death mediated by
the immune system. As such, the hydrogel micro-carriers described
herein permit encapsulation and administration of heterologous
cells, e.g., without triggering adverse immune responses.
[0163] In some examples, the stiffness of the hydrogel
microparticles, e.g., alginate microparticles, is controlled by
altering initial divalent cation (e.g., calcium carbonate
nanoparticle) concentrations, changing polymer (e.g., alginate)
concentration, and/or treating the microparticles with additional
divalent cations (e.g., calcium ions) after formation. In some
cases, to characterize the stiffness, fluorescently labeled
microparticles are adhered to a coated glass surface (e.g.,
poly-1-lysine coated glass surface) or a polydimethylsiloxane
(PDMS) microwell, and probed by atomic force microscopy (AFM) to
measure Young's Modulus (in Pa) (FIG. 7).
[0164] To support cell adhesion to the microparticles, the polymer
(e.g., alginate) is optionally conjugated with functional ligands,
e.g., by carbodiimide chemistry, with varying degrees of
conjugation (substitution) to control ligand density. Exemplary
ligands include an Arg-Gly-Asp (RGD) peptide (which binds to
integrins .alpha.V, .alpha.5.beta.1, .alpha.8.beta.1, and
.alpha.IIb.beta.3) and a Leu-Asp-Val (LDV) peptide (which binds to
integrins .alpha.4.beta.1, .alpha.4.beta.7, .alpha.9.beta.1,
.alpha.E.beta.7, and the .beta.2 subfamily) (Humphries et al.,
2006, J Cell Sci 119, 3901-3903).
[0165] For example, to test whether hydrogel microparticles can
endure shear force experienced by blood circulation after
intravenous injection, they were subjected to controlled in vitro
shear by a rheometer. Hydrogel (e.g., alginate) microparticles
described herein with a Young's Modulus of about 1 kPa (which is in
a stiffness range comparable to that of some blood cell types)
remained intact after shear force beyond the physiological arterial
wall stress (about 2 Pa, FIG. 8), demonstrating that the particles
would be able to remain intact in the blood circulation.
The Compositions of the Invention Comprising Hydrogel Capsules
Exhibit Prolonged In Vivo Biodistribution Kinetics
[0166] In some cases, cells encapsulated in hydrogel capsules and
administered in vivo remain viable for a longer period of time than
cells not encapsulated in the hydrogel capsules, e.g., cells
directly injected/infused into a patient. For example, the
encapsulated cell(s) comprise a longer serum/plasma stability or
halflife compared to non-encapsulated cell(s). For example,
hydrogel encapsulated cells remain viable after administration in
vivo for at least 6 hours, e.g., at least 6, 12, 24, 48 hours, 1,
2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, 1, 2, 3, 4, 5, or 6
months, or longer. In other examples, encapsulated cells remain at
the site of a targeted tissue (e.g., if injected/infused directly
into a target tissue (e.g., lung, heart, kidney, skin, bone marrow)
for a longer period of time than non-encapsulated cells. For
example, encapsulated cells remain at the site of a targeted tissue
for at least 1 hour (e.g., at least 1, 3, 6, 12, 24, 48 hours, 1,
2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5, 6 weeks, 1, 2, 3, 4, 5, or 6
months, or longer). Because of the prolonged biodistribution of the
delivered cells, the cell-containing hydrogel micro-carriers also
circumvent the need for more frequent administrations of cells or
cell-secreted factors to a patient.
[0167] For example, the hydrogel micro-carriers described herein
also increase the cell count, e.g., by physically shielding the
cell from immune cells or other clearance mechanisms by the body,
and/or by increasing the growth rate of the cell. Exemplary cells
included in the micro-carriers comprise MSCs, HSCs or progenitors
thereof, or endothelial progenitors. In some cases, the hydrogel
micro-carriers comprise a stiffness, e.g., that maintains or
increases the cell count compared to other stiffnesses.
[0168] To track in vivo biodistribution kinetics of injected cells
in hydrogel microparticles by live imaging (e.g. IVIS, Perkin
Elmer), the hydrogel is conjugated with fluorescent dyes (e.g.
Alexa 750) by carbodiimide chemistry, or with biotin first,
followed by binding streptavidin-conjugated fluorescent dyes. For
example, cells are labeled with vital dyes (DiR) or are genetically
engineered to express fluorescent (e.g. mCherry) or luminescent
(e.g. luciferase) proteins. Firefly luciferase is used, e.g., to
track the localization of the injected cells, while Gaussia
luciferase is used, e.g., as a surrogate marker for proteins that
are secreted from the transplanted cells into biological fluids,
including blood and urine (Tannous, 2009, Nat Protoc 4,
582-591).
[0169] In some embodiments, once cells are encapsulated in hydrogel
microparticles, they are injected into the body via different
administration routes. Exemplary routes include intravenous
(retro-orbital, tail-vein), intraperitoneal, subcutaneous,
intracardial, intrabone, intratracheal, intramuscular, topical, and
oral.
[0170] Retro-orbital injection of a solution containing fluorescent
dye-conjugated alginate hydrogel into mice (C57BL/6J) showed
sustained accumulation of alginate in the body for more than 2
weeks without compromising health, indicating that alginate is
biocompatible (FIG. 9). As shown in FIG. 9, cell-free alginate
microparticles with a diameter of 30 .mu.m were produced by a
microfluidic method as described above, and injected intravenously.
The particles remained in lungs for at least 1 week after
intravenous delivery. Particle diameter was about 25-30 .mu.m, and
particle stiffness was about 0.3 to 1 kPa. Microparticles were
injected retroorbitally (left side). More particles were observed
in the left lung at day 0 (P<0.03) than at day 1. At day 1, the
signal in the left lung was decreased and that in the right lung
was increased. The signals in both the left and right lungs were
then decreased over time after more than 1 week.
[0171] As shown in FIG. 10, mouse (D1) mesenchymal stem cells
(MSCs) were overexpressed with mCherry and firefly luciferase. They
were then encapsulated in alginate microparticles conjugated with
Alexa 750. An amount of 10.sup.5 MSCs per mouse was injected. After
retro-orbital injection, both luciferase and Alexa 750 signals were
tracked for 1 week. While directly injected cells were cleared in
24 hours, the cells encapsulated in alginate microparticles lasted
longer in lungs than free cells that were not encapsulated, e.g.,
by greater than 2-fold (e.g., greater than 2, 3, 4, 5, 6, 7, 8, 9,
or 10-fold, e.g., greater than 10-fold), as indicated by the
calculation of the area under curve.
[0172] Further, to confirm that that prolonged biodistribution of
MSCs in lungs after intravenous injection was due to physical
shielding of single cells rather than mixing with alginate, cells
in alginate microparticles were compared with unencapsulated cells
that were mixed with empty alginate microparticles. The results
showed that unencapsulated cells mixed with empty alginate beads
were cleared within 24 hours, while encapsulated cells were alive
and remained in lungs at this 24 hours (e.g., at least 24 hours,
e.g., at least 24, 36, 48 hours or greater) (FIG. 11).
[0173] In addition, as shown in FIG. 12, alginate capsules without
cells were localized in the liver when they were injected directly
into the left ventricle of the heart. This was in contrast to the
retro-orbital injection, where the particles were localized
predominantly in the lungs.
[0174] Further, experiments were done with human MSCs that express
mCherry and luciferase. After encapsulation, human MSCs were
injected intravenously in NOD/SCID/IL2gamma.sup.-/- mice that were
sublethally irradiated (2.5 Gy). The results showed a similar trend
as those from the experiments with mouse cells described above,
demonstrating prolonged biodistribution after encapsulation (FIG.
13).
[0175] In the hydrogel capsules of the invention, physical
properties of the hydrogel encapsulating the cells may be altered.
Changes in the physical properties of the hydrogel may, in turn,
alter the biological effect that the hydrogel capsules exert on a
subject being administered the compositions of the invention. For
example, changing the stiffness of the hydrogel may alter secretion
of protein factors, e.g., hematopoietic factors, from an
encapsulated cell, e.g., an MSC. For example, FIG. 14 demonstrates
the effect of altering physical properties of the hydrogel on the
number of hematopoietic stem cells (HSC) and committed progenitors
(CPP). Specifically, physical properties of alginate hydrogels were
altered either by changing concentration of calcium used for the
cross-linking reaction; by varying alginate concentration; or by
varying alginate molecular weight ("10/60": 120 kDa or "20/40": 250
kDa). The physical properties of the resulting hydrogels were
characterized using a rheometer. Soft hydrogels that comprised
HSCs/progenitors were more effective at increasing the number of
HSCs, as compared to stiffer hydrogels. Accordingly, by varying the
stiffness/composition of the hydrogels, the number of HSCs and
progenitors is maximized in vivo by encapsulating the
HSCs/progenitors in soft alginate microparticles followed by
injection.
[0176] For example, hydrogel microparticles may comprise a cell,
e.g., an HSC or an CPP. Such hydrogel microparticles are
characterized by low stiffness, e.g., have an elastic modulus of
about 1-5000 Pa, e.g., 10-1000 Pa, e.g., 10-500 Pa, e.g., 20-300
Pa, e.g., 30-300 Pa, e.g., 50-300 Pa, e.g., 50-150 Pa. Optionally,
such hydrogels comprises a divalent cation (e.g., calcium ion) at
the concentration of about 1-10 mM, e.g., 1-5 mM, e.g., 1-2 mM,
and, e.g., an alginate molecular weight of about 120 kDa.
[0177] In some examples, the physical properties of the hydrogel
capsules control the release kinetics of secretions from
encapsulated cells. For example, as shown in FIG. 15, there was an
optimal stiffness for alginate hydrogels to control the release of
secretory factors from MSCs, which in turn maximized the number of
HSCs. Hydrogel stiffness increased the total amount of proteins
released per cell (e.g., MSC). The release kinetic profiles of
individual proteins in response to hydrogel stiffness was distinct
depending on the size of the proteins.
[0178] Given the prolonged biodistribution of in vivo injected
single cells encapsulated in hydrogel microparticles (FIGS. 10-13),
the results show that the mechanics of the hydrogels can be
harnessed to control the release of proteins from the encapsulated
single cells in vivo.
[0179] This approach is not limited to the delivery of endogenously
secreted factors. In some examples, cells are engineered to
overexpress exogenous (e.g., recombinant) secretory proteins (e.g.
Gaussia Luciferase), and their release kinetics are controlled by
hydrogel microparticles that encapsulate the cells (and their
stiffness).
[0180] In some cases, to control the release of secreted factors
from cells, the pore size of alginate microparticles is varied.
Alginate hydrogels without any modification are nanoporous (<5
nm pore size), permitting the passage of proteins up to the size of
50 kDa (e.g. Gaussia Luciferase, which is .about.19 kDa). However,
in some cases, hydrogels are modified to comprise pores of other
sizes, e.g., >5 nm, e.g., at least 10 nm, e.g., 10-500 nm, e.g.,
10-100 nm.
[0181] Tuning the stiffness of hydrogels induces protein release
from cell, e.g., MSCs. Proteins can be secreted directly into the
extracellular space via conventional exocytosis pathways (e.g.,
direct release without extracellular vesicles). In other examples,
proteins can also be released indirectly, e.g., by packaging into
extracellular vesicles that include exosomes (40.about.100 nm in
diameter) and microvesicles (0.1-1 .mu.m). For example, these
vesicles are also known to contain small RNAs, such as microRNAs.
Directly released proteins are capable of leaving a hydrogel
micro-carrier comprising pores of 5 nm or less, e.g, alginate-based
hydrogel micro-carrier (which comprise a pore size of 5 nm or
less). However, for those proteins that are released indirectly via
extracellular vesicles (e.g, size/diameter >50 nm), the vesicles
cannot leave the hydrogel micro-carrier on their own (FIG. 15). To
allow the release of the vesicles, the pore size of the hydrogel
micro-carrier is, e.g., increased, e.g., to hundreds of nm or more.
For example, hematopoietic or other factors (e.g., those that aid
in recovery of damaged heart tissue) are secreted directly or via
extracellular vehicles.
[0182] In some examples, a hydrogel described herein, e.g.,
alginate hydrogel, does not allow the passage of an extracellular
vesicle (FIG. 15). In some cases, to enable the release of
extracellular vesicles from hydrogel capsules, rapidly degradable
nanoparticles with a defined size are incorporated when the
hydrogel capsules are synthesized, e.g., to generate pores in the
hydrogel capsules. The materials appropriate for degradable
nanoparticles include, e.g., oxidized alginate and poly
(D,L-lactide-co-glycolide) (PLGA). Sub-micron porous hydrogel
micro-carriers are also useful for the delivery of synthetic
nanoparticles, such as gold and magnetic nanoparticles.
[0183] In some examples, to permit the release of cell-derived
microparticles, e.g., exosomes, microvesicles, or other parts of a
cell, that are larger than nanoparticles but smaller than the size
of a cell (e.g., between 1 and 10 .mu.m), monodisperse spherical
porogens with a size (e.g., diameter) between 1 and 10 .mu.m are
microfluidically formed as described above, e.g., with oxidized
alginate. In some cases, the porogens are then encapsulated along
with cells in larger hydrogel microparticles, and they are degraded
to form pores in the hydrogel microparticles. Naturally occurring
cell-derived microparticles that are delivered by the method
include platelets and apoptotic bodies. These particles are
directly incorporated into the microparticles or produced by
encapsulating their precursors, including megakaryocytes and
apoptotic cells, respectively.
[0184] In some examples, pore size of the hydrogel capsules is
controlled such that certain secreted molecules are released from
the hydrogel, e.g., into the rest of the body, and other secreted
molecules are trapped inside the hydrogel, e.g., kept from
diffusing into the rest of the body. For example, nanoporous
hydrogels (e.g., alginate hydrogels) comprise a pore size of 20 nm
or less (e.g., 20, 15, 10, 5, 4, 3, 2, 1 nm or less). Such pore
sizes permit the migration, e.g., outward migration, of proteins of
about 200 kDa or less (e.g., 200, 150, 100, 90, 80, 70, 60, 50, 40,
30, 20, 10, 5, 2 kDa or less) through the pores. Such pore sizes
exclude the migration, e.g., outward migration, of larger
molecules, e.g., with a molecular weight of greater than 200 kDa,
e.g., exosomes, microvesicles, or other microparticles (such as
platelets or apoptotic bodies) through pores. In other examples the
hydrogel micro-carrier comprises larger pores. For example, larger
pores, e.g., comprising a diameter of greater than 20 nm (e.g., 30
nm, or greater, 30, 40, 50, 60, 80, 100, 120, 150, 200, 400, 600,
800 nm, 1 .mu.m, 2 .mu.m, 4 .mu.m, 8 .mu.m, or greater) permit the
migration of secreted proteins as well as secreted exosomes,
microvesicles, and microparticles (e.g., platelets and apoptotic
bodies) through the pores, e.g., out of the hydrogel.
[0185] In accordance with the hydrogels and methods described
herein, prolonged in vivo delivery of MSCs improves the treatment
of a range of disease conditions. Cell therapies using MSCs are
currently tested for over 300 clinical trials, but the
biodistribution kinetics of MSCs after intravenous injection is
generally too short, as shown in FIGS. 10 and 13. Prolonging the
biodistribution of MSCs by single cell encapsulation, as provided
by the methods herein, is thus useful for treating a number of
disease conditions where MSCs have been shown to be beneficial. For
example, MSCs have been shown to be useful in the treatment of
degenerative, inflammatory, and autoimmune diseases. See, e.g.,
Farini et al. Stem Cells Intl. 2014, 2014:306573. MSCs have
immunomodulatory effects, e.g., they inhibit cytotoxic T cells and
natural killer (NK) cells, e.g, by secreting suppressors of T-cell
development (such as TGF-beta and hepatocyte growth factor (HGF))
and suppressors of proliferation (such as leukemia inhibitory
factor (LIF) and interferon-gamma (IFN-gamma)). MSCs can also
induce expression of tumor necrosis factor-alpha (TNF-alpha) and
interleukin-1 (IL-1), which can lead to secretion of chemokines and
inducible nitric oxide synthase (iNOS). See id. Also, MSCs have
been used in the treatment of musculoskeletal diseases, such as
Duchenne muscular dystrophy (DMD), as well as in the regeneration
of muscle, e.g., skeletal muscle tissue, and the treatment of
osteonecrosis, spinal fusion disease, severe osteogenesis
imperfect. See id. MSCs are also useful in the treatment of
cardiovascular diseases/cardiovascular repair, as MSCs secrete
molecules that have important effects on the cellular
microenvironment and also differentiate into cardiomyocytes. MSCs
decrease fibrosis in the myocardium and improve myocardial
contractility and ventricular function, and are useful for treating
dilated cardiomyopathy. See id. For example, molecules secreted by
MSCs are able to protect the myocardium by preserving its
contractile capacity; in particular, MSCs-derived cytokines inhibit
the apoptosis of cardiomyocytes, allowing the formation of new
vessels in damaged tissues. See id. MSCs are also useful for
treating liver diseases, e.g., cirrhosis, end-stage liver disease,
and fulminant hepatic failure (FHF), e.g., by reducing
hepatocellular death and increasing hepatocellular proliferation.
See id. In addition, MSCs have been used to block the development
of chronic inflammatory processes that occur in autoimmune
arthritis, diabetes, and lupus. Accordingly, MSCs are useful for
the treatment of rheumatoid arthritis, systemic lupus
erythematosus, and Type 1 diabetes. In other cases, MSCs are useful
for treating neurodegenerative diseases, such as amyotrophic
lateral sclerosis, multiple sclerosis, Alzheimer's disease, and
Parkinson's disease. See id.
[0186] The single cell encapsulation methods and hydrogels
described herein are useful for improving the secretion of
therapeutic factors from the encapsulated MSC and/or the
integration of the delivered MSC into the host. Disease conditions
treatable using the hydrogels/methods described herein include
hematological malignancies, bone marrow failure, bone marrow
transplantation, graft versus host disease (GVHD), acute radiation
syndrome, cardiac regeneration, acute respiratory distress
syndrome, and septic shock (Syed and Evans, 2013, Nat Rev Drug
Discov 12, 185-186). For example, MSCs suppress T-cell activation
under some conditions in vitro (Di Nicola et al., 2002, Blood 99,
3838-3843), but the efficacy of MSCs is likely to be diminished in
vivo due to the rapid clearance after injection. Single cell
encapsulation is thus useful to improve the efficacy of MSCs to
prolong the suppression of donor T-cells that cause GVHD in
patients that undergo hematopoietic transplantation, e.g., HSC
transplantation.
[0187] For example, a MSC described herein, e.g., encapsulated in a
hydrogel described herein, secretes a cardiovascular regeneration
factor and/or a GVHD suppression factor. Cardiovascular
regeneration factors from MSCs include vascular endothelial growth
factor (VEGF), stromal cell derived factor 1 (SDF-1), tumor
necrosis factor-inducible gene 6 protein (TSG-6), interleukin-6
(IL-6), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF
or FGF-2), insulin-like growth factor 1 (IGF-1), hepatocyte growth
factor (HGF), Thrombospondin-4, secreted frizzled-related protein 2
(Sfrp2), matrix metalloproteinase 9 (MMP-9), tissue inhibitor of
metalloproteinases (TIMP) metallopeptidase inhibitor 2 (TIMP-2),
monocyte chemotactic protein 1 (MCP-1), thrombospondin 1 (TSP-1),
chemokine (C-X-C motif) ligand 6 (CXCL6), interferon gamma-induced
protein 10 (IP-10). For examples, Genbank Accession Nos. for these
protein factors are incorporated herein by reference and are as
follows: VEGF (P15692.2), SDF-1 (P48061.1), TSG-6 (P98066.2), IL-6
(P05231.1), IL-8 (P10145.1), FGF-2 (P09038.3), IGF-1 (CAA01955.1 or
CAA01954.1), HGF (P14210.2), thrombospondin-4 (NP 003239.2), Sfrp2
(Q96HF1.2), MMP-9 (P14780.3), TIMP-2 (P16035.2), MCP-1 (P13500.1),
TSP-1 (Q9HCB6.2 or NP 003237.2), CXCL6 (P80162.4), and IP-10
(P02778.2).
[0188] GVHD suppression factors from MSCs include transforming
growth factor-beta (TGF-beta), hepatocyte growth factor (HGF),
prostaglandin E2 (PGE2), galectin, and Indoleamine 2,3-dioxygenase
(IDO). For examples, Genbank Accession Nos. for the protein factors
are incorporated herein by reference and are as follows: TGF-beta
(AAA36738.1), HGF (P14210.2), galectin (88922.1), and IDO
(P14902.1). The structure of PGE2 is shown below:
##STR00002##
[0189] In addition, prolonged in vivo delivery of endothelial
progenitors is useful for improving treatment of a number of
diseases. For example, endothelial progenitors are delivered via
single cell encapsulation methods as described herein to improve
the regeneration of tissues in vivo. Tissues to be regenerated by
endothelial progenitors include blood, damaged muscles, liver, and
lungs (Manayski et al., 2014, Circ Res 114, 1077-1079).
[0190] Further, prolonged delivery of engineered cells and
microorganisms is applicable to approaches in synthetic biology.
For example, cells and microorganisms are engineered to express a
genetic circuit that can respond to specific factors known to be
present in physiological and pathological conditions (Wieland and
Fussenegger, 2012, Annu Rev Chem Biomol Eng 3, 209-234). The single
encapsulation method described herein permits the in vivo
application of the synthetic biology approach by improving the
biodistribution kinetics of the engineered cells and microorganisms
that can sense and respond to signals from their milieu. From a
safety perspective, because alginate microparticles without
modification have a small pore size (<5 nm), they can be used to
physically shield engineered microorganisms within the host after
transplantation, while still permitting the passage of therapeutic
molecules from the engineered microorganisms into the host.
II. Methods of Preparing Hydrogel Capsules Containing Cells
[0191] High-throughput encapsulation of cells into microscale
hydrogels is a technology that has great utility and promise but
that currently involves several challenges. For example, challenges
include achieving a thin hydrogel layer around encapsulated cells,
increasing the yield of cell-containing particles, controlling the
mechanical properties of the hydrogel, and enabling long-term
culture of encapsulated cells. The present invention provides a
gentle method for single-cell encapsulation that may comprise the
following steps:
[0192] a) contacting a cell with a moiety capable of adhering to a
cell and comprising a cross-linking catalyst; and
[0193] b) contacting the cell and the moiety with at least one
polymer comprising a plurality of polymer chains;
wherein the cross-linking catalyst catalyzes a reaction that
cross-links said plurality of polymer chains. In certain examples,
the moiety capable of adhering to a cell and comprising a
cross-linking catalyst may be a nanoparticle, such as a CaCO.sub.2
nanoparticle, a BaCO.sub.3 nanoparticle, or a SrCO.sub.3
nanoparticle.
[0194] Without wishing to be bound by a specific theory, it is
believed that adherence of the moiety comprising a cross-linking
catalyst to a cell prior to hydrogel formation is important for
increasing encapsulation yield, i.e., fraction of all hydrogel
capsules in the composition that comprise a cell, e.g., one or more
cells. It is also believed, without wishing to be bound by a
specific theory, that adherence of the moiety comprising a
cross-linking catalyst to a cell prior to hydrogel formation is
important for increasing encapsulation efficiency, i.e., fraction
of all cells in the composition that become encapsulated.
Accordingly, adherence of a moiety comprising a cross-linking
catalyst to a cell prior to hydrogel, allows for producing
compositions with at least 90% encapsulation yield and at least 90%
efficiency.
[0195] In specific examples provided herein, single-cell
encapsulation in alginate is achieved by adsorbing calcium
carbonate nanoparticles to cells prior to forming cell droplets.
The method was tested on three cell types, a mesenchymal stem cell
line (D1), a preadipocyte cell line (OP9), and primary outgrowth
endothelial cells (OEC). Depending on the cell type, the efficiency
and yield of encapsulation ranged from 49% to 88%, and the
viability over a three-day period ranged from 60% to 90%. Using
Dis, the hydrogel capsules were mechanically tractable, and hybrid
hydrogel capsules were successfully formed from a mixture of
polymers. Also, a PDMS microwell culture system was used to study
osteogenic differentiation of encapsulated mesenchymal stem
cells.
[0196] The methods described herein overcome many of the previous
challenges with high-throughput encapsulation of cells into
hydrogels. Encapsulation of single cells by a thin hydrogel layer
is useful for a variety of fields, including the assembly of
complex tissues, high throughput small molecule and drug screens,
and cell delivery therapies. Microfluidics and surfactant chemistry
have been used to encapsulate cells in microscale hydrogels (>60
.mu.m), but these approaches suffer several drawbacks. For example,
previously described hydrogel capsules are generally much larger
than the cells they encapsulate, and, in most approaches,
increasing the fraction of droplets containing cells requires high
cell densities and often results in multiple cells per droplet.
See, e.g., Selimovic et al. Polymers (Basel). 4, 1554 (2013); Chung
et al. Lab Chip. 12, 45-59 (2012); Martinez et al. Macromol.
Biosci. 12, 946-951 (2012); and Tan et al. Adv Mat. 19, 2696-2701
(2007). Little work has been done on controlling hydrogel
properties and on long-term culture of cells encapsulated in
hydrogel capsules.
[0197] The methods and compositions described herein provide
advancements to the field of single-cell encapsulation, including
cell encapsulation within a thin hydrogel layer; a one-step method
for increasing the fraction of hydrogel capsules containing cells;
control over mechanical properties of the hydrogel matrix;
fabrication of cell-encapsulating hybrid hydrogel capsules; and
demonstration that assembled encapsulated cells can function
analogously to cells in 2D at similar size scales.
[0198] Provided herein is a method for encapsulating cells in a
thin hydrogel coating and creating hydrogel capsules comprising
cells. The method achieves high yield, i.e., results in composition
that comprises a high fraction of hydrogel capsules that contain a
cell, e.g., one or more cells. The method also achieves high
efficiency, i.e., results in a high fraction of cells that are
encapsulated into hydrogel capsules. The encapsulated cells
produced by the method of the invention are also characterized by
high long-term viability. Further, physical properties of the
hydrogel encapsulating the cells may be altered, thereby
controlling the behavior of microencapsulated cells.
[0199] In some embodiments, the present invention provides a
hydrogel capsule comprising a polymer and 50 or fewer cells (e.g.,
50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer, e.g., 1
cell), where the hydrogel capsule comprises a diameter of 500 .mu.m
or less (e.g., 500, 400, 300, 200, 150, 100, 90, 80, 70, 60, 50,
40, 30, 20 .mu.m or less), and where the cells are fully surrounded
by a layer of hydrogel. In other embodiments, the hydrogel capsule
bead diameter is at least 20 .mu.m (e.g., at least 20, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 .mu.m, or greater).
For example, the diameter is 20-500 .mu.m. For example, the layer
of hydrogel surrounding the cells is 0.5 to 20 .mu.m thick (e.g.,
0.5-15 .mu.m, 0.5-12 .mu.m, 0.5-10 .mu.m, 0.5-7.5 .mu.m, 1-20
.mu.m, 1-15 .mu.m, 1-10 .mu.m, 1-5 .mu.m, 0.8-12 .mu.m, or about
4.8 .mu.m thick). As used herein, the term "diameter", when used in
reference to the hydrogel capsule having an irregular spherical
shape, refers to the longest dimension of the hydrogel capsule. The
term "diameter", as used herein, also refers to the diameter of a
hydrogel capsule having a perfect spherical shape.
[0200] Polymers include biocompatible polymers, e.g., that can be
cross-linked in a cell-compatible, gentle, way. In the presence of
calcium, the polymer forms a solid, and its elastic modulus is
adjusted by changing the concentration of its cross-linker. An
exemplary suitable polymers include alginate, collagen, fibrin,
agarose, poly(ethylene glycol dimethacrylate), polylactic acid,
polyglycolic acid, PLGA, gelatin, agarose, poly(lysine),
polyhydroxybutyrate, poly-epsilon-caprolactone, polyphosphazines,
poly(vinyl alcohol), poly(alkylene oxide), poly(ethylene oxide),
poly(allylamine), poly(acrylate), poly(4-aminomethylstyrene),
pluronic polyol, polyoxamer, poly(uronic acid), poly(anhydride) or
poly(vinylpyrrolidone). In one embodiment, the polymer is
alginate.
[0201] For example, the polymer (e.g., alginate) is ionically
crosslinked by a divalent cation, such as Ca.sup.2+, Mg.sup.2+,
Sr.sup.2+, Ba.sup.2+, Be.sup.2+ or Al.sup.2+. In one embodiment,
the divalent cation comprises Ca.sup.2+. In one embodiment,
alginate is used in an encapsulating hydrogel. Alginate is a
biocompatible polymer capable of a gentle mode of cross-linking. In
the presence of a divalent ion (e.g., calcium), alginate crosslinks
to form a hydrogel, and it elastic modulus can be adjusted by
changing the concentration of the divalent ion (e.g., calcium).
[0202] A multitude of cell types are suitable for encapsulation
using the methods described herein. For example, a cell may be a
mesenchymal stem cell (MSC) or a progenitor thereof, a
hematopoietic stem cell (HSC) or a progenitor thereof,
pre-adipocyte cell, or an endothelial cell.
[0203] In the studies described herein, cells were pre-coated with
cation-containing nanoparticles (e.g., calcium carbonate), mixed
with liquid polymer (e.g., sodium alginate), and extruded through a
small aperture within a microfluidic device. Dissolution of the
nanoparticles led to polymer (e.g., alginate) cross-linking,
producing a thin hydrogel layer around encapsulated cells. Because
nanoparticles containing the cation crosslinker were adsorbed to
the cells prior to cross-linking, the method achieved a high
fraction of cell-containing hydrogel capsules.
[0204] Accordingly, a method of encapsulating a cell in a hydrogel
capsule may comprises the following steps:
[0205] a) providing a microfluidic device comprising intersecting
open channels;
[0206] b) providing an aqueous phase liquid comprising a cell, a
divalent cation or salt thereof, and a polymer;
[0207] c) providing a nonaqueous phase liquid comprising an oil and
an acid;
[0208] d) injecting the aqueous phase liquid into a channel of the
device while simultaneously injecting the nonaqueous phase into a
separate channel of the device to form an emulsion; and
[0209] e) contacting the emulsion with a cell-compatible solution
to form an individual polymeric hydrogel capsule bead comprising a
cell encapsulated within the hydrogel capsule.
[0210] In some embodiments, the aqueous phase liquid of step b) is
made by the following steps:
[0211] mixing cells with nanoparticles comprising the divalent
cation or salt thereof, thereby coating the cells with the
nanoparticles;
[0212] removing unbound nanoparticles from mixture; and
[0213] mixing the nanoparticle coated cells with the polymer.
[0214] For example, the nanoparticle, e.g., comprising a divalent
cation or salt thereof, comprises a diameter of 400 nm to 1000 nm
(e.g., 450-950 nm, 500-900 nm, 400-800 nm, 500-900 nm, 550-800 nm,
600-800 nm, 550-750 nm, or about 680 nm). In some examples, the
nanoparticle is present at a concentration of 1-50 mg/mL or 50 mM
or less (e.g., 1-40 mg/mL, 1-30 mg/mL, 1-20 mg/mL, 1-10 mg/mL, 5-50
mg/mL, 10-50 mg/mL, 20-50 mg/mL, 30-50 mg/mL, 40-50 mg/mL, 20-40
mg/mL, 20-30 mg/mL, 30-40 mg/mL; or 40 mM, 30 mM, 25 mM, 20 mM, 15
mM, 10 mM, 5 mM, 2.5 mM, 1.5 mM, 1 mM or less).
[0215] In some examples, a nanoparticle described herein comprises
a zeta potential in media of -10 mV or lower (more negative), e.g.,
-10 mV to -60 mV, e.g., about -20 mV, for example, -23 mV. The
magnitude of (absolute value of) the zeta potential is a measure of
the degree of electrostatic repulsion between adjacent, similarly
charged particles in a dispersion. For example, a high zeta
potential (negative or positive) indicates that the solution or
dispersion is more electrically stabilized and will resist
aggregation, while a low zeta potential (negative or positive)
indicates that the dispersion is more likely to break and aggregate
or flocculate.
[0216] In some cases, the aqueous phase liquid comprises a divalent
cation at a concentration of 1-50 mg/mL or 50 mM or less (e.g.,
1-40 mg/mL, 1-30 mg/mL, 1-20 mg/mL, 1-10 mg/mL, 5-50 mg/mL, 10-50
mg/mL, 20-50 mg/mL, 30-50 mg/mL, 40-50 mg/mL, 20-40 mg/mL, 20-30
mg/mL, 30-40 mg/mL; or 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5
mM, 2.5 mM, 1.5 mM, 1 mM or less).
[0217] In some embodiments, the divalent cation is Ca.sup.2+,
Mg.sup.2+, Sr.sup.2+, Ba.sup.2+, or Be.sup.2+. For example, the
divalent cation is Ca.sup.2+. In other embodiments, the trivalent
cation comprises Al.sup.3+.
[0218] In some cases, the oil of step c) of the method is a
fluorinated oil, soybean oil, corn oil, mineral oil, hexadecane, or
a combination thereof. Exemplary acids suitable for the method
include acetic acid, formic acid, benzoic acid, oxalic acid, lactic
acid, propionic acid, butyric acid, and combinations thereof.
[0219] In some examples, the nonaqueous phase liquid further
comprises a surfactant.
[0220] In some embodiments, a cell compatible solution, e.g., as
used in step e) of the method, includes cell culture media.
[0221] In accordance with the methods described herein, the
channels microfluidic device comprise a diameter of 10-500 .mu.m
(e.g., 10-400 .mu.m, 10-300 .mu.m, 10-250 .mu.m, 10-200 .mu.m,
10-150 .mu.m, 10-100 .mu.m, 10-80 .mu.m, 10-60 .mu.m, 10-40 .mu.m,
20-500 .mu.m, 40-500 .mu.m, 60-500 .mu.m, 80-500 .mu.m, 100-500
.mu.m, 150-500 .mu.m, 200-500 .mu.m, 250-500 .mu.m, 300-500 .mu.m,
400-500 .mu.m, 100-400 .mu.m, 100-300 .mu.m, 200-400 .mu.m, or
200-300 .mu.m).
[0222] In some examples, the hydrogel capsule bead comprises a
diameter of 10-500 .mu.m (e.g., 10-400 .mu.m, 10-300 .mu.m, 10-250
.mu.m, 10-200 .mu.m, 10-150 .mu.m, 10-100 .mu.m, 10-80 .mu.m, 10-60
.mu.m, 10-40 .mu.m, 20-500 .mu.m, 40-500 .mu.m, 60-500 .mu.m,
80-500 .mu.m, 100-500 .mu.m, 150-500 .mu.m, 200-500 .mu.m, 250-500
.mu.m, 300-500 .mu.m, 400-500 .mu.m, 100-400 .mu.m, 100-300 .mu.m,
200-400 .mu.m, or 200-300 .mu.m). For example, the hydrogel capsule
bead diameter is at least 20 .mu.m (e.g., at least 20, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 .mu.m, or
greater).
[0223] In some embodiments, to encapsulate single cells in hydrogel
capsules, e.g., with a diameter ranging from 10 to 100 .mu.m,
calcium (or other divalent or trivalent cations) carbonate
nanoparticles are adsorbed to cell membrane, washed out, and mixed
with alginate (or other crosslinkable biomaterial) solutions. For
example, the mixture is then passed through a microfluidic device
to form an emulsion comprising microparticles that contain cells.
The hydrophobic phase contains oil, e.g., fluorocarbon oil, mixed
with a surfactant and an acid, e.g., acetic acid. The acid, e.g.,
acetic acid, releases the divalent cation from the salt, e.g.,
carbonate, thereby ionically crosslinking alginate in the presence
of cells. In some cases, a chemical such as perfluoro-1-octanol is
then used to break the emulsion and isolate the aqueous phase that
contains single cells encapsulated in hydrogel microparticles. This
method may yield about 50% of hydrogel capsules that contain single
cells with the cell viability after encapsulation of at least 10%,
e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or higher. For
example, as shown in FIG. 7, this method may yield cell viability
after encapsulation of about 50%, based on flow cytometry
analysis.
[0224] In some cases, before encapsulation, the polymer, e.g.,
alginate, agarose, or gelatin, is mixed with naturally occurring
extracellular matrix proteins, such as collagen, fibronectin,
and/or laminin.
[0225] In accordance with the method, 50 or fewer cells (e.g., 50,
40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer cells) are
encapsulated in the hydrogel capsule bead. In one embodiment, one
cell is encapsulated in the hydrogel capsule bead. Exemplary cells
include a mesenchymal stem cell (MSC), a pre-adipocyte cell, or an
endothelial cell. Multiple different cell types are suitable for
encapsulation by the methods described herein. For example, the
encapsulation yield, efficiency, and long-term viability of
mesenchymal stem cells (MSCs) (e.g., D1s, which are a murine MSC
line), pre-adipocyte cells (which support differentiation of
embryonic stem cells into hematopoietic stem cells), and human
outgrowth endothelial cells (OECs) are described in the
Examples.
[0226] As shown in the results herein, cells were encapsulated in
hydrogel capsules were as small as about 30 microns in diameter,
such that the hydrogel layer around encapsulated cells averaged
about 4.8 .mu.m in thickness. The thickness was as thin as about
0.8 .mu.m in some areas. There was no region in which the hydrogel
layer around the cell was absent. This is equal in size to
cell-encapsulating hydrogel capsules fabricated in a
high-throughput manner (see, e.g., Park et al. Lab Chip
14(2014):1873-1879), and roughly half the size of the smallest
hydrogel capsules demonstrated thus far with non-cancerous cells
(see, e.g., Martinez et al. Macromol. Biosci. 12, 946-951 (2012)).
The hydrogel capsules presented here are also smaller than those
reported using photolithography, the smallest of which are 50 .mu.m
in diameter (see, e.g., Liu et al. Sci. China. Chem 55, 494-501
(2012)). The smaller hydrogel capsule size provided by the
invention is beneficial in a number of ways. Constructs assembled
from cells encapsulated in smaller hydrogel capsules have a higher
maximal cell density. The thinner gel layer improves diffusion of
nutrients to the cells as well as signaling and communication
between cells. Also, smaller hydrogel capsule sizes make cell
injection therapies easier and more feasible by permitting
encapsulated cells to enter blood vessels of smaller diameter.
[0227] As described in the Examples, the variation in polymer
content of cell-encapsulating hydrogel capsules was assessed by
flow cytometry, and hydrogel capsule size variation was assessed by
microscopy. The variability in empty hydrogel capsule size was
similar to values obtained in previous studies on the fabrication
of monodisperse beads and hydrogel capsules. As unencapsulated
cells were found to have a CV in diameter of 0.23, it is likely
that greater variation in polymer content and size in
cell-encapsulating hydrogel capsules were due to the presence of
cells. Cell-encapsulating hydrogel capsules exhibited a
quasi-monodisperse size distribution (see, e.g., de la Vega et al.
Nanomedicine (Lond). 8.2(2013):265-85). This finding demonstrates
that encapsulated cells are exposed to similar extracellular
environments, which is useful for studies assessing cell-cell
variability on a single cell level.
[0228] As described in the Examples, a high encapsulation yield was
achieved by pre-coating cells with divalent cation nanoparticles
(e.g., calcium carbonate nanoparticles). The exact yield varied
with cell type, but was consistently an order of magnitude higher
than the yield following direct injection of non-coated cells into
a microfluidic device.
[0229] Encapsulation of cells into droplets has been found to
follow a Poisson distribution,
P(k)=.lamda..sup.ke.sup.-.lamda./k!
where P(k) is the fraction of droplets expected to contain k cells,
and .lamda. is the average number of cells per drop, which is
affected by droplet size and cell density. The actual fraction of
hydrogel capsules with D1 cells encapsulated by direct injection
(0.073) was slightly lower than the expected value predicted with a
Poisson process (0.087). Encapsulation yields of OECs (0.028) and
OP9s (0.066) with the direct injection process were slightly higher
than those predicted by a Poisson process (0.024 and 0.057,
respectively). See, e.g., FIGS. 18E-F. Direct injection
encapsulation yields were similar to Poisson distribution
prediction. This demonstrates that most alginate droplets
crosslinked and that the encapsulation yield was determined by
droplet size and cell density. However, by preventing the
cross-linking of non-cell containing droplets via the cation
nanoparticle pre-coating step, the fraction of hydrogel capsules
containing cells was greatly enriched without increasing hydrogel
capsule size or cell density. See, e.g., FIG. 18A-F. Few studies
have been conducted with the aim of improving encapsulation yield
either in liquid droplets or in hydrogels, and attempts to address
this issue in the past utilized two-step processes (see, e.g., Wu
et al. Biomed Microdevices 15, 553-560 (2013)) or low-throughput
approaches. The methods provided herein are advantageous for
approaches or compositions that require a pure population of
cell-encapsulating hydrogel capsules.
[0230] The impact of polymer molecular weight (MW) on cell
behavior, e.g., cell division and egress from encapsulating
hydrogel capsules, is described herein. Different MW of polymer
(e.g., alginate) were used to modulate cell division within and
cell egress from hydrogel capsules. Exemplary MW of polymer (e.g.,
alginate) suitable for use in accordance with the compositions and
methods described herein include MW of at least 50 kDa (e.g., at
least 50, 75, 100, 125, 150, 175, 200, 230, 250, 260, 275, 300,
350, 400, 450, 500, or greater). Other MWs of polymers are, e.g.,
50-500 kDa (e.g., 50-400 kDa, 50-300 kDa, 50-250 kDa, 50-200 kDa,
50-150 kDa, 50-100 kDa, 100-500 kDa, 100-400 kDa, 100-300 kDa, or
100-250 kDa). For example, the MW of a polymer (e.g., alginate) is
54 kDa, 139 kDa, or 232 kDa.
[0231] Higher MW polymers (e.g., alginates) decreased cell egress
from and cell division within each hydrogel capsule bead.
Mechanical testing revealed that higher MW (e.g., about 232 kDa)
polymer (e.g., alginate) hydrogel capsules had a greater elastic
modulus than hydrogel capsules fabricated from lower MW polymer
(e.g., alginate). This finding shows that the stiffer matrix may
have prevented cell-mediated remodeling of the matrix required to
make space for cell division and migration. The elastic moduli of
hydrogel capsules described herein were an order of magnitude lower
than that of large, bulk hydrogels with the same composition.
Moreover, the bulk elastic moduli of the same alginate hydrogels
were found to be higher than those obtained through AFM. In some
cases, this finding is due to the mechanical behaviors of alginate
hydrogels differing on different size scales. In other cases, as
the AFM probe is of similar dimensions to pores in the alginate
network, a larger tip may be needed to accurately measure alginate
hydrogels. The increase of the elastic moduli of cell-encapsulating
hydrogel capsules with increasing calcium concentration and
increasing polymer weight shows that ECM properties were
manipulated in hydrogel capsules to control the behavior of
encapsulated cells.
[0232] As extracellular matrix (ECM) mechanical properties have
been shown to strongly influence cell behavior, modulating hydrogel
capsule mechanical properties, e.g., by changing the polymer MW,
provides a means to control the behavior of encapsulated cells. In
some examples, divalent cation (e.g., calcium) treatments were used
to alter matrix mechanics after fabrication. For example, cation
(e.g., calcium) concentration is used to further modulate the
elastic moduli of cell-encapsulating hydrogel capsules after the
encapsulation process. For example, the results described herein
show that addition of divalent cation (e.g., calcium) to the
cell-encapsulated hydrogel capsules after the hydrogel capsule
formation increased the elastic moduli. See, e.g., FIG. 18L.
[0233] Since hydrogels composed of proteins native to the body,
e.g., collagen and fibrin, possess properties lacking in alginate
alone, the fabrication of collagen-alginate and fibrin-alginate
hybrid hydrogel capsules using the methods described herein was
also developed. As described in the Examples, cell-encapsulating
hybrid hydrogel capsules made of collagen and alginate, or fibrin
and alginate, were fabricated. The two components of each hydrogel
capsule (e.g., alginate and collagen, or alginate and fibrin) were
evenly distributed within/among hydrogel capsules, and the
resultant hydrogel capsules supported cells in long-term culture.
Some phase separation of the alginate in fibrin-alginate hydrogel
capsules was observed, likely due to the natural calcium-binding
properties of fibrinogen. The alginate polymers were evenly
distributed on a larger size scale. Greater variation in hydrogel
capsule composition was observed in fibrin-alginate hydrogel
capsules than collagen-alginate hydrogel capsules. This is likely
due to the rapid fibrin cross-linking at the junction of the two
aqueous phases affecting the laminar flow of the two polymer
components. The fibrin-alginate hydrogel capsules were also
significantly poorer in maintaining cell viability, likely due to
fibrin formation being insufficiently rapid to prevent cell
exposure to the oil-surfactant phase. Consistently, cells
encapsulated in fibrin-only hydrogel capsules, which were made with
a higher concentration of thrombin, showed higher viability. The
ability to fabricate cell-encapsulating hydrogel capsules of
different polymers and different combinations of polymers expands
the potential applications of hydrogel capsules, for example to
wound healing therapies (see, e.g., Gorodetsky et al. J. Invest.
Dermatol. 112, 866-872 (1999)). Hybrid hydrogel capsules combine
the properties of two types of polymers. Because the protein
network (e.g., collagen or fibrin network) of hybrid hydrogel
capsules maintains structural integrity to some extent
independently of the alginate portion, the hybrid hydrogel capsules
are advantageous in that they permit switching of the cell
microenvironment from hybrid to unitary using protein digestion
(e.g., to digest the protein network) or divalent ion (e.g.,
calcium) chelation (e.g., to remove the alginate crosslinks).
[0234] Accordingly, also provided herein is a composition
comprising hybrid hydrogel capsules, wherein the hydrogel comprises
a first polymer and a second polymer, wherein each of the hybrid
hydrogel capsules comprise 50 cells or fewer, where the microgel
comprises a diameter or longest dimension of 500 .mu.m or less, and
where the cells are fully surrounded by a layer of hydrogel.
[0235] In some embodiments, the first polymer is a polysaccharide,
e.g., alginate. In some cases, the second polymer is a protein,
e.g., collagen or fibrin.
[0236] A method for making hybrid microgels may involve the
following steps:
[0237] a) providing a microfluidic device comprising intersecting
open channels;
[0238] b) mixing a divalent cation nanoparticle with cells, a first
polymer (e.g., polysaccharide, such as alginate), and a second
polymer (e.g., protein, such as collagen or fibrin) to produce an
aqueous phase liquid;
[0239] c) providing a nonaqueous phase liquid comprising an oil and
an acid;
[0240] d) injecting the aqueous phase liquid into a channel of the
device while simultaneously injecting the nonaqueous phase into a
separate channel of the device to form an emulsion; and
[0241] e) contacting the emulsion with a cell-compatible solution
to form an individual polymeric microgel bead comprising a cell
encapsulated within the microgel.
[0242] In some embodiments, the method further comprises a step of
incubating the aqueous phase liquid and nonaqueous phase in the
device as an emulsion for a specified period of time (e.g., at
least 15 s, e.g., at least 15 s, 30 s, 1 min, 2 min, 4 min, 6 min,
10 min, 20 min, 30 min, 45 min, 60 min, 1.5 h, or more) before
contacting the emulsion with a cell-compatible solution. In some
cases, step d) of the method is performed at a temperature of about
30-40.degree. C., e.g., about 30.degree. C., 31.degree. C.,
32.degree. C., 33.degree. C., 34.degree. C., 35.degree. C.,
36.degree. C., 37.degree. C., 38.degree. C., 39.degree. C., or
40.degree. C.
[0243] In some examples, the second polymer is present in a
neutral-pH liquid form.
[0244] In some examples, the injection step b) is performed at a
temperature of 10.degree. C. or less (e.g., 10, 8, 6, 4.degree. C.,
or less).
[0245] As discussed in the Examples, the osteogenic differentiation
potential of encapsulated cells (e.g., D1 MSCs) assembled in a
microwell-containing device (e.g., PDMS microwells) was tested.
PDMS microwells were capable of templating and culturing singly
encapsulated cells. The resultant assemblages had similar densities
across microwell sizes. Deeper wells prevented sample loss from the
larger-diameter microwells during washing and media change steps.
Two-dimensional micropatterning studies have linked increased
homotypic cell-cell contact in MSCs with increased differentiation
(Tang et al. Biomaterials 31, 2470-76 (2010); and Wang et al. J
Biomed Mater Res Part A. 101A(12), 3388-95 (2013)).
[0246] A comparable relationship between cell number and
differentiation was found in the microwell assemblages in the
results herein. The use of microwells to assemble and culture
encapsulated cells improves techniques for fabricating microscale
tissues. Although bioprinting techniques can pattern single cells
with high spatial resolution on a two-dimensional (2-D) surface,
cells cultured in 2-D do not always behave in a way that accurately
reflects their behavior in native 3D environments. Printing of
hydrogel encapsulated cells, for example, can combine high spatial
resolution with a more relevant 3D environment. Moreover, as the
PDMS microwells remain open to addition of other cells or soluble
factors, the microwell-templated assemblages are a useful platform
for co-culture experiments with timed entries, e.g., for drug
screens.
[0247] Accordingly, the invention also provides a device comprising
a microwell, where the microwell comprises a hydrogel capsule or
hybrid hydrogel capsule described herein. For example, the
microwell comprises a diameter of 25 .mu.m to 500 .mu.m. In other
examples, the microwell comprises a surface area of about 400,000
.mu.m.sup.2 to about 600,000 .mu.m.sup.2 (e.g, 400,000-550,000
.mu.m, 400,000-500,000 .mu.m, 450,000-600,000 .mu.m,
500,000-600,000 .mu.m.sup.2). In some cases, the microwell
comprises a depth of at least 100 .mu.m (e.g., at least 100 .mu.m,
150 .mu.m, 200 .mu.m, 250 .mu.m, 300 .mu.m, 350 .mu.m, 400 .mu.m,
450 .mu.m, 500 .mu.m or greater). For example, the microwell
comprises 1 to 50 cells, e.g., 1-40, 1-35, 2-50, 2-40, 2-37, 2-35,
2-30, 3-50, 3-40, 3-37, 3-35, 3-30, 5-50, 5-40, 5-35, 5-30, 10-50,
10-40, 10-35, or 10-30 cells.
[0248] In some embodiments, the device comprises
polydimethylsiloxane (PDMS). For example, the device is useful as a
template for cell culture or tissue growth, e.g., microscale tissue
fabrication.
III. Methods of Using Hydrogel Capsules Containing Cells
[0249] The present invention provides methods for delivering cells
and protein factors that may be secreted by the cells, to a subject
in need thereof, as well as methods for treating subjects, e.g.,
subjects having a cardiovascular disease or an immunological
disorder, using the hydrogel capsules of the invention.
[0250] Delivering cells and their secretions to the body remains a
major challenge due to rapid clearance of exogenous cells by
physical and immune barriers (Yoo et al., 2011, Nat Rev Drug Discov
10, 521-535). The ability to control the shielding of exogenously
administered cells from the body's natural clearance mechanism can
potentially lead to significant improvement in the in vivo delivery
of cells and biologics that the cells produce. While a bulk
crosslinked hydrogel has been used for this purpose (Nicodemus and
Bryant, 2008, Tissue Eng Part B Rev 14, 149-165), it is difficult
to inject the bulk gel into the body, and the administration route
is generally limited to subcutaneous. The compositions of the
invention comprising hydrogel capsules overcome this challenge, as
the hydrogel capsules formed from a crosslinked hydrogel
encapsulate individual cells so that they can be delivered in the
body using a wide range of injection routes. In addition to
improving cell delivery, physical and chemical properties of the
hydrogel capsules can be altered to direct cellular behavior in
vivo for diagnostic and therapeutic purposes.
[0251] Since the first human bone marrow transplantation (BMT)
(Thomas et al., 1957, N Engl J Med 257, 491-496) and the revelation
of hematopoietic stem cell/progenitors (HSC/Ps) by clonal assay
(Becker et al., 1963, Nature 197, 452-454), HSC/Ps have become an
intense focus of research to improve sustained in vivo regeneration
and ex vivo production of blood for patients. Lamins have been
described to regulate cell trafficking and lineage maturation of
adult human hematopoietic cells. See, e.g., Shin et al. Proc. Natl.
Acad. Sci., 2013 Nov. 19; 110(47):18892-7, incorporated herein by
reference. Contractile forces have been shown to sustain and
polarize hematopoiesis from stem and progenitor cells. See, e.g.,
Shin et al. Cell Stem Cell. 14, 1-13 (2014), incorporated herein by
reference. Also, myosin-II inhibition and soft 2D matrices have
been shown to maximize multinucleation and cellular projections
typical of platelet-producing megakaryocytes. See, e.g., Shin et
al. Proc. Natl. Acad. Sci. 2011 Jul. 12; 108(28):11458-63,
incorporated herein by reference. The bone marrow (BM) `niche` or
`microenvironment` was proposed to be a basic unit that regulates
HSC self-renewal and differentiation (Schofield, 1978, Blood Cells
4, 7-25). It has been suggested that multipotent BM mesenchymal
stromal cells (MSCs) provide key regulatory signals to program
hematopoiesis, based on studies with cultures derived from the
adherent fraction of the BM stroma (Dexter et al., 1977, J Cell
Physiol 91, 335-344; Friedenstein et al., 1976, Exp Hematol 4,
267-274). A number of studies explored the ability of this 2D
stromal culture system or isolated growth factors from the stroma
to expand HSC/Ps ex vivo, but no methods have been described that
are sufficient to preserve long-term repopulating HSCs and to
achieve clinically relevant effects (Broxmeyer, 2011, Cell Prolif
44 Suppl 1, 55-59).
[0252] Limitations of using MSCs to facilitate clinical blood
production are in part due to at least two issues. First, MSCs are
not well defined at a clonal level. For example, MSCs need to be
better defined at a clonal level using a serial transplantation
assay to assess their capability of forming a new BM niche upon
subcutaneous implantation (Bianco et al., 2013, Nat Med 19, 35-42).
Recent studies revealed human BM MSCs as CD146+(Sacchetti et al.,
2007, Cell 131, 324-336) and Nestin+ (Pinho et al., 2013, J Exp
Med. 210(7): 1351-1367) cells. These purified cells expanded HSC/Ps
ex vivo in a paracrine manner (Isern et al., 2013, Cell Rep 3,
17141724). Second, rigid plastic dishes have been used in many past
studies, such dishes do not reflect the native BM milieu, which may
provide necessary cues for MSCs to secrete appropriate
hematopoietic factors. Cells generate contractile forces via
actomyosin, and they pull on and respond to the mechanical
properties of the substrates they are grown on, followed by the
activation of mechano-sensitive transcription factors (Discher et
al., 2005, Science 310, 1139-1143). The in situ BM exhibits
heterogeneous matrix stiffness measured in Young's modulus (E,
unit: Pa). The central marrow is generally soft (<0.3 kPa),
while the osteoid is rigid (.about.1,000 kPa), but much less than
plastic (.about.GPa) (Shin et al., 2014, Cell Stem Cell 14, 81-93).
Using hydrogels with tunable physical properties, MSCs specify
their lineages based in part on the stiffness of the substrate they
are grown on in both 2D (Engler et al., 2006, Cell 126, 677-689)
and 3D (Huebsch et al., 2010, Nat Mater 9, 518-526).
[0253] Cells secrete molecules either directly into the
extracellular space or through small extracellular vesicles, which
are made up of exosomes (40-200 nm in diameter) and/or
microvesicles (.about.1 .mu.m or greater in diameter) (Raposo and
Stoorvogel, 2013, J Cell Biol 200, 373-383) or other types of
microparticles (such as platelets and apoptotic bodies). For
example, microvesicles are vesicles derived from cells, e.g.,
containing biological material. For example, microvesicles are at
least 1 .mu.m in diameter (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,
or 9 .mu.m) and are, e.g., smaller than a cell (e.g., 10 .mu.m or
less, e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2 .mu.m, or less).
Microparticles, e.g., platelets, are about 1-7 .mu.m in diameter,
e.g., 1, 2, 3, 4, 5, 6, or 7 .mu.m. In the hematopoietic system,
recent studies described that exosomes released from MSCs impact
blood cancer cell proliferation (Roccaro et al., 2013, J Clin
Invest 123, 1542-1555), but their roles in normal hematopoiesis
remain unknown. In some cases, actomyosin contractile forces drive
the final factor release step at the cell membrane (Masedunskas et
al., 2011, Proc Natl Acad Sci USA 108, 13552-13557;
Muralidharan-Chari et al., 2009, Curr Biol 19, 1875-1885). Cells
pulling on the stiff substrate experience high plasma membrane
tension generated by actomyosin forces (Engler et al., 2006, Cell
126, 677-689), and increased membrane tension activates exocytosis
(Gauthier et al., 2011, Proc Natl Acad Sci USA 108, 14467-14472).
However, prior to this disclosure, it was unclear whether substrate
stiffness controls factor secretion from MSCs.
[0254] Human bone marrow (BM) generates 10.sup.5 to 10.sup.6
nucleated blood cells, a nucleated red blood cells, and platelets
every second. A single stem cell can generate the entire blood
hierarchically, and this ability is clinically important to improve
therapies with hematopoietic stem cell and progenitors (HSC/Ps).
HSC/Ps are regulated by BM niches, which are made up of mesenchymal
stromal cells (MSCs) and their lineages. MSCs generate force
through cytoskeletons, engage extracellular matrix (ECM), and sense
the local matrix stiffness. Matrix stiffness regulates lineage
differentiation and migration of MSCs, but the role of matrix
stiffness in other force-dependent biological functions has been
unclear. MSCs secrete various soluble factors that support
hematopoiesis, and cytoskeletal forces regulate exocytosis. The
present invention provides methods to harness matrix stiffness,
e.g., of hydrogels used to encapsulate cells, to control secretion
of soluble factors from MSCs, which in turn, e.g., influences
hematopoiesis in a paracrine manner.
[0255] The stiffness of the hydrogels encapsulating cells in the
compostions of the invention may be altered to regulate release of
protein factors secreted from the cells. For example, the hydrogel
stiffness may be altered to regulate secretion of hematopoietic
factors from MSCs that may regulate hematopoiesis. To induce
mechanical triggering of paracrine release from MSCs in vivo and
sustainably deliver factors that impact BM resident HSC/Ps in vivo,
a microfluidics method is described herein that encapsulates
individual cells, e.g., MSCs, in micro-hydrogel droplets so that
they can be infused in an intravenous or intrabone route (FIG.
1).
[0256] The stiffness and viscoelasticity of materials, such as the
hydrogels described herein, are determined by applying a stress
(e.g., oscillatory force) to the material and measuring the
resulting displacement (i.e., strain). For an applied oscillatory
stress or strain, the stress and strain occur in phase in purely
elastic materials, such that the response of one (stress or strain)
occurs simultaneously with the other. In purely viscous materials,
a phase difference is detected between stress and strain. The
strain lags behind the stress by a 90 degree (radian) phase lag.
Viscoelastic materials have behavior in between that of purely
elastic and purely viscous--they exhibit some phase lag in strain.
The storage modulus in viscoelastic solid materials are a measure
of the stored energy, representing the elastic portion, while the
loss modulus in viscoelastic solids measure the energy dissipated
as heat, representing the viscous portion. In some examples, the
hydrogels described herein are characterized as viscoelastic.
[0257] The elastic modulus (also called Young's modulus) is a
measure of stiffness of a material, such as a hydrogel. The elastic
modulus is the slope of the initial straight portion, e.g., the
first 5-10% of strain, of a stress-strain curve. The modulus is the
ratio of the change in stress to the change in strain expressed as
a fraction of the original length. The elastic modulus has units of
Pa (or N/m.sup.2 or m.sup.-1kgs.sup.-2). For viscoelastic
materials, the measured elastic modulus can depend on the timescale
of the stress-strain measurement, since viscoelastic materials can
exhibit stress relaxation leading to a decrease in the measured
modulus for measurements taken over longer timescales. For
materials that exhibit significant stress relaxation, the initial
elastic modulus is defined as the elastic modulus for a
stress-strain measurement that is performed over a timescale at
which there is minimal stress relaxation. The initial elastic
modulus can be determined using standard methods available in the
art, e.g., by a compression test or rheology. For example, a
hydrogel is compressed, e.g., to 15% strain, e.g., with a
deformation rate of about 1 mm/min. With 15% compression, the
stress versus strain relations of the hydrogels are almost linear,
and the slope of the initial portion (first 5-10% strain) of the
stress strain curves gives the initial elastic modulus.
[0258] For example, the stiffness of a hydrogel described herein
can be tuned/modulated over the range of typical tissues, e.g.,
from liquid (blood) to soft tissues to harder tissues (e.g., bone).
For example, the stiffness of a hydrogel can be tuned over the
range of soft tissues (heart, lung, kidney, liver, muscle, neural,
etc.) from an elastic modulus of .about.20 Pascals (fat) to 100,000
Pascals (skeletal muscle). Bone marrow is about 300 Pa. An osteoid
is about 34 kPa. Different tissue types are characterized by
different stiffness, e.g., normal brain tissue has a shear modulus
of approximately 200 Pascal. For example, blood is characterized by
stiffnesses of less than 1 kPa. Brain tissue is characterized by
stiffnesses of about 1 kPa. Muscle has a stiffness of about 10 kPa.
Collagenous bone has a stiffness of about 100 kPa. Blood, bone
marrow, and neuronal cells have a physiological stiffness of about
0.1 kPa to about 1 kPa, adipose cells have a physiological
stiffness of about 0.1 kPa to about 3 kPa, liver, kidney, fat,
lung, and endothelial cells have a physiological stiffness of about
1 kPa to about 10 kPa, muscle and heart cells have a physiological
stiffness of about 10 kPa to about 20 kPa, and cartilage and bone
cells have a physiological stiffness of about 20 kPa to about 500
kPa. For example, a hydrogel described herein comprises a stiffness
that matches that of the normal stiffness of a cell/tissue type
described above, e.g., a cell type that is encapsulated into the
hydrogel.
[0259] BM MSCs secrete molecules not only directly to the
extracellular space but also through vesicles. Effects of matrix
stiffness on the extracellular vesicle release from MSCs are
described herein. For example, matrix stiffness affects the
quantity of vesicles released from MSCs and/or the content of the
vesicles released from MSCs. In some cases, the quantity and
content of these vesicles impacts HSC/P differentiation, e.g., in
vitro and/or in vivo. For example, the protein quantity and
contents of the vesicles and the conditioned media from MSC
cultures are determined. In some examples, mechano-sensitive
transcription is required for matrix stiffness-dependent release of
soluble factors. In some cases, RNA interference against
Yes-associated protein and serum response factor in MSCs is used to
determine whether mechano-sensitive transcription is required for
the matrix stiffness-dependent release of the soluble factors. In
addition, small molecules against the myosin pathway are used to
determine the involvement of contractile forces in the stiffness
dependent release of soluble factors. Exemplary small molecules
that inhibit the myosin pathway include (+/-)-blebbistatin (EMD
Biosciences), reversine, ML-7, and Y-27632 (Sigma).
[0260] In some examples, the invention also provides a method of
promoting secretion of a protein factor, e.g., a hematopoietic
factor, from a cell (e.g., a MSC, HSC or progenitor thereof, or
endothelial progenitor) through release of vesicles, comprising
contacting the cell with a hydrogel micro-carrier described herein.
In other examples, the invention provides a method of promoting
secretion of a protein factor, e.g., a hematopoietic factor, from a
cell (e.g., an MSC, HSC or progenitor thereof, or endothelial
progenitor) through direct secretion (e.g., without exosome release
into an extracellular space), comprising contacting the cell with a
hydrogel micro-carrier described herein. In some examples, the
invention provides a method of preferentially promoting secretion
of a hematopoietic factor from a mesenchymal stem cell (MSC)
through release of vesicles as opposed to other mechanisms of
secretion (e.g., direct secretion), comprising contacting the MSC
with a hydrogel micro-carrier described herein. In some examples,
the vesicles comprise a diameter of at least about 1 .mu.m, e.g.,
at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 40, 60, 80, or
100 .mu.m. In other examples, the vesicles comprise a diameter of
1000 nm or less (e.g., 1000, 900, 800, 500, 250, 150, 100, 80, 60,
50, 40, 30, 20, 10 nm or less).
[0261] The invention is based in part on the discovery of how
matrix mechanics regulate paracrine release. This knowledge, in
addition to the biomaterials and microtechnologies described
herein, are used to develop an infusible micro-vesicle that can
mechanically trigger sustainable factor secretion from cells, e.g.,
MSCs. MSCs secrete factors, including factors that treat heart
damage/disease and factors that regulate hematopoiesis. In one
example, the methods and hydrogels described herein produce
mechano-sensitive factors from MSCs capable of programming
hematopoiesis both ex vivo and in vivo. The hydrogel capsules and
methods described herein are also applicable to the treatment and
prevention of hematopoietic and cardiovascular diseases. For
example, the hydrogel capsules and methods described herein are
useful for the improvement of hematopoietic engraftment, e.g., for
bone marrow transplantation, and the exploration of the MSC
secretome is applicable to the treatment of cardiovascular and
other diseases. In some cases, MSCs release proteins that are known
to treat myocardial infarction, hypertrophy, and arrhythmia. For
example, such factors include cardiovascular regeneration factors,
e.g., vascular endothelial growth factor (VEGF), stromal cell
derived factor 1 (SDF-1), tumor necrosis factor-inducible gene 6
protein (TSG-6), interleukin-6 (IL-6), interleukin-8 (IL-8), basic
fibroblast growth factor (bFGF or FGF-2), insulin-like growth
factor 1 (IGF-1), hepatocyte growth factor (HGF), Thrombospondin-4,
secreted frizzled-related protein 2 (Sfrp2), matrix
metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinases
(TIMP) metallopeptidase inhibitor 2 (TIMP-2), monocyte chemotactic
protein 1 (MCP-1), thrombospondin 1 (TSP-1), chemokine (C-X-C
motif) ligand 6 (CXCL6), or interferon gamma-induced protein 10
(IP-10).
[0262] In some examples, an MSC is included in/on a hydrogel
micro-carrier and administered to a subject intravenously or
directly into a tissue, e.g., a heart tissue. For example, by
including, e.g., encapsulating, a MSC in a hydrogel micro-carrier
and directly injecting the micro-carrier into a heart tissue, the
factors from MSCs are secreted in a prolonged and controlled manner
to treat heart conditions.
[0263] The present invention provides infusible hydrogel capsules
that mechanically trigger hematopoietic factor release from
encapsulated MSCs in vivo. MSCs likely secrete a number of factors
that act together to regulate hematopoiesis. This invention
harnesses these mechano-sensitive secretory factors to program BM
resident HSC/Ps in vivo by using a microfluidics-based technique to
achieve single cell encapsulation of MSCs. In particular, alginate
gel droplets, e.g., with a size appropriate for intravenous and
intrabone infusion, are fabricated by varying cross-linker (e.g.,
divalent cation, e.g., Ca') and gel concentrations to achieve
optimal mechanical properties for MSCs to induce HSC expansion. The
stiffness and size of the droplets are varied in order to optimize
in vivo clearance kinetics of the droplets after infusion. In some
cases, factors released from MSCs, e.g., the MSCs encapsulated in
the hydrogel droplets described herein, enhance human
hematopoiesis. For example, such enhancement of human hematopoiesis
is detected by transplantation of immunocompromised xenograft mice
with human BM CD34.sup.+ cells, followed by infusion of MSC
droplets and standard characterization of human hematopoietic
reconstitution.
[0264] In some cases, the hydrogel capsules and methods described
herein are useful for generation of blood, e.g., for transfusions.
There is a need for adequate supplies of blood components,
including red blood cells (RBCs). Methods have been described for
producing mature human RBCs having the characteristics of native
adult RBCs from hematopoietic stem cells of diverse origins, e.g.,
blood, bone marrow, or cord blood. See, e.g., Douay et al.
Transfus. Med. Rev. 2007, 21(2):91-100. The invention provides a
method of generating RBCs in vitro/ex vivo by using matrix
stiffness-modulated MSCs that stimulate hematopoiesis by HSCs by
paracrine signaling between MSCs and HSCs. For example, the
invention includes a method comprising providing a hydrogel
comprising a MSC and a stiffness, e.g., where the stiffness of the
hydrogel promotes secretion of hematopoietic factors from the MSC.
In some examples, the stiffness preferentially promotes secretion
of hematopoietic factors via release of vesicles as opposed to
other mechanisms of secretion. The method optionally comprises a
step of collecting an extracellular content, e.g., secreted
substance, from the MSC. The method further comprises contacting a
secreted substance from the MSC with a HSC. In some cases, the
hydrogel comprising a MSC is located in the same closed container
as the HSC, e.g., such that a secreted substance from the MSC is
capable of coming into contact with the HSC, e.g., by diffusion
through a liquid (e.g., culture medium), e.g., via diffusion
through a pore of a membrane that separates the MSC from the HSC.
For example, the method preserves long-term repopulating HSCs that
are capable of producing blood cells, e.g., RBCs. For examples, the
method increases the number of HSCs in culture, e.g., by promoting
the growth rate and/or survival of HSCs.
[0265] In some embodiments, the invention also provides a method of
enhancing secretion of a hematopoietic factor by a MSC by
contacting the MSC with a 2-dimensional or 3-dimensional hydrogel
described herein, e.g., comprising a stiffness that enhances
secretion of the hematopoietic factor by the MSC.
[0266] Secretion is the process for releasing and/or oozing a
substance, e.g., a chemical or biological substance/molecule, from
a cell or gland. Several mechanisms exist for secretion of
biological substances, e.g., proteins, from cells. For example,
proteins targeted for secretion into the extracellular space of a
cell are translated at the rough endoplasmic reticulum (ER), where
they are glycosylated, folded, and shuttled into the Golgi
apparatus. In the Golgi, glycosylation of the protein is modified
and posttranslational modifications occur. The proteins to be
secreted are shuttled into secretory vesicles that travel to the
cell membrane, where the vesicle fuses with the cell membrane in a
process called exocytosis. In some types of secretion, the contents
of the vesicle, e.g., including secreted proteins, are dumped into
the extracellular space. In other types of secretion, proteins are
secreted by release of exosomes/vesicles containing the protein(s)
from a cell. This process involves endosomes themselves
invaginating their membrane. As the invaginations break off, they
produce vesicles within vesicles, called multivesicular bodies.
When these vesicles fuse with the cell's plasma membrane, these
tiny (e.g., 40-100 nm) internal vesicles--called exosomes are
secreted. Yet other mechanisms of secretion can involve, e.g.,
direct translocation of a protein across a cell membrane, e.g.,
through transporters in the membrane. Another secretion mechanism
is lysosomal secretion. Yet another secretion mechanism involves
release of molecules, e.g., proteins, from cells by mechanical or
physiological wounding, e.g., through nonlethal, transient pores in
the cell membrane.
[0267] The secretion of protein factors from a cell occurs by
exosomal release or by direct secretion of the protein factor into
an extracellular space of the cell, e.g., an MSC. In some cases,
the hydrogel encapsulating cells in the compositions of the
invention promotes exosomal release of the hematopoietic factor by
the MSC. In other cases, the hydrogel promotes direct secretion of
the hematopoietic factor into an extracellular space of the MSC. In
some examples, the hydrogel preferentially promotes exosomal
release as opposed to direct secretion of the hematopoietic factor
by the MSC. In other examples, the hydrogel preferentially promotes
direct secretion of the hematopoietic factor as opposed to exosomal
release of the factor.
[0268] For example, the secretion occurs by exosomal release or by
direct secretion of the hematopoietic factor into an extracellular
space of the MSC. In some cases, the hydrogel promotes exosomal
release of the hematopoietic factor by the MSC. In other cases, the
hydrogel promotes direct secretion of the hematopoietic factor into
an extracellular space of the MSC. In some examples, the hydrogel
preferentially promotes exosomal release as opposed to direct
secretion of the hematopoietic factor by the MSC. In other
examples, the hydrogel preferentially promotes direct secretion of
the hematopoietic factor as opposed to exosomal release of the
factor.
[0269] The invention further features a method of enhancing HSC
engraftment following bone marrow transplantation or HSC
transplantation in a subject by administering to the subject a
2-dimensional or 3-dimensional hydrogel described herein, e.g.,
comprising a stiffness and a MSC.
[0270] Bone marrow is the soft, spongy tissue found inside bones
that comprises stem cells, e.g., hematopoietic stem cells (HSCs).
Bone marrow is the medium for development and storage of most of
the body's blood cells.
[0271] Bone marrow transplant (BMT) is a therapy used for patients
suffering from diseases such as cancer, immunodeficiency disorders,
and blood disorders. In some examples, a BMT taking cells normally
found in bone marrow (e.g., stem cells), filtering those cells, and
administering them to the patient (e.g., where the patient is the
same or different person as the donor). In some examples, the BMT
transfuses healthy bone marrow cells into a patient after his or
her bone marrow has been entirely or partially destroyed by
treatment that kills abnormal cells in the patient's body. Such
treatments can include cancer therapies, e.g., chemotherapy,
radiation, and/or cancer vaccines. For example, BMT has been used
to effectively treat diseases such as leukemias, lymphomas,
aplastic anemia, immune deficiency disorders, and some solid tumor
cancers.
[0272] Exemplary cancers include a melanoma, a central nervous
system (CNS) cancer, a CNS germ cell tumor, a lung cancer,
leukemia, multiple myeloma, a renal cancer, a malignant glioma, a
medulloblatoma, a breast cancer, an ovarian cancer, a prostate
cancer, a bladder cancer, a fibrosarcoma, a pancreatic cancer, a
gastric cancer, a head and neck cancer, or a colorectal cancer. For
example, a cancer cell is derived from a solid tumor cancer or
hematological/blood cancer. The hematological cancer is, e.g., a
leukemia or a lymphoma. A leukemia is acute lymphoblastic leukemia
(ALL), acute myelogenous leukemia (AML), chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma (SLL), chronic
myelogenous leukemia (CML), or acute monocytic leukemia (AMoL). A
lymphoma is follicular lymphoma, Hodgkin's lymphoma (e.g., Nodular
sclerosing subtype, mixed-cellularity subtype, lymphocyte-rich
subtype, or lymphocyte depleted subtype), or Non-Hodgkin's
lymphoma. Exemplary solid cancers include but are not limited to
melanoma (e.g., unresectable, metastatic melanoma), renal cancer
(e.g., renal cell carcinoma), prostate cancer (e.g., metastatic
castration resistant prostate cancer), ovarian cancer (e.g.,
epithelial ovarian cancer, such as metastatic epithelial ovarian
cancer), breast cancer (e.g., triple negative breast cancer), and
lung cancer (e.g., non-small cell lung cancer).
[0273] In some examples, a BMT or HSC transplant is useful for
cures a disease, e.g., a type of cancer or blood disorder. In other
examples, a BMT or HSC transplant is useful for replenishing a
damaged bone marrow after a treatment, e.g., chemotherapy or
radiation. For example, the doses of chemotherapy or radiation
required to cure a cancer are so high that a person's bone marrow
stem cells are sometimes permanently damaged or destroyed by the
treatment. Other times, a person's bone marrow can be destroyed by
a disease.
[0274] For example, a bone marrow transplant or HSC transplant is
used to replace diseased, nonfunctioning bone marrow/HSCs with
healthy functioning bone marrow/HSCs, e.g., for conditions such as
leukemia, aplastic anemia, or sickle cell anemia. BMT or HSC
transplant is also used to regenerate a new immune system in order
to fight existing or residual leukemia or other cancers not killed
by the chemotherapy or radiation used in the transplant. In
addition, BMT or HSC transplant is used to replace the bone marrow
and restore its normal function after high doses of chemotherapy
and/or radiation are given to treat a malignancy. This process is
often called rescue (e.g., for diseases such as lymphoma and
neuroblastoma). Also, BMT or HSC transplant is used to replace bone
marrow with genetically healthy functioning bone marrow to prevent
further damage from a genetic disease process (such as Hurler's
syndrome and adrenoleukodystrophy).
[0275] Types of BMT include autologous bone marrow transplant,
allogeneic bone marrow transplant, or umbilical cord blood
transplant. In autologous BMT, the donor is the patient himself or
herself. Stem cells are taken from the patient either by bone
marrow harvest or apheresis (a process of collecting peripheral
blood stem cells), frozen, and then administered to the patient
after a treatment. In allogeneic BMT, the donor shares the same
genetic type as the patient. Stem cells are taken either by bone
marrow harvest or apheresis from a genetically matched donor, e.g.,
a relative, e.g., a brother or sister. Other donors for allogeneic
bone marrow transplants can include a parent or an unrelated donor,
e.g., found through national bone marrow registries. In umbilical
cord blood transplant, stem cells are taken from an umbilical cord
immediately after delivery of an infant. These stem cells reproduce
into mature, functioning blood cells quicker and more effectively
than do stem cells taken from the bone marrow of another child or
adult. The stem cells are tested, typed, counted, and frozen until
they are needed for a transplant. A HSC transplant can also involve
an autologous HSC or allogeneic HSC.
[0276] After BMT/HSC transplant, engraftment occurs, in which the
donated cells travel to the bone marrow of the patient and begin
producing new blood cells. In some examples, engraftment can be
delayed because of infection, medications, low donated stem cell
count, or graft failure. New bone marrow may begin making cells in
the first 30 days following transplant, but it may take months,
even years, for the entire immune system to fully recover after a
BMT/HSC transplant. Sometimes, complications occur after transplant
that can delay engraftment. Exemplary complications include
infections, low platelets (thrombocytopenia) and low red blood
cells (anemia), pain (e.g., related to mouth sores and
gastrointestinal irritation), fluid overload (e.g. which can lead
to pneumonia, liver damage, and high blood pressure), respiratory
distress, organ damage (e.g., liver or heart damage), graft failure
(e.g., failure of the graft (transplant) to take hold in the
marrow), and Graft-versus-host disease (GVHD) (e.g., where the
donor's immune system reacts against the recipient's
tissue/cells).
[0277] In some cases, the hydrogel capsules and methods of the
invention mitigate one or more of these complications associated
with BMT or HSC transplantation. In other cases, the hydrogels and
methods of the invention enhance engraftment of stem cells (e.g.,
from a bone marrow or HSC transplant), e.g., decrease the time for
engraftment after transplantation.
[0278] As used herein, autologous refers to donor cells that are
provided by the patient himself/herself. Allogeneic refers to donor
cells that are of the same species but genetically non-identical to
the patient. Related allogeneic refers to cells provided by
patient's sibling or other family member. Unrelated allogeneic
refers to cells provided by a volunteer donor, e.g., who is not the
patient's family member.
[0279] A hydrogel is a gel comprising interconnected crosslinked
polymer strands. For example, a hydrogel comprises pores, e.g.,
that can hold passenger molecules/cells. In such a way, the
hydrogel can serve as a carrier/delivery vehicle for
molecules/cells. A hydrogel micro-carrier is a hydrogel for
delivery a passenger molecule/cell that is small enough to be
injected/infused into a subject, e.g., through a blood stream of
the subject. For example, a hydrogel micro-carrier carries a small
number of cells, e.g., 50 or fewer, e.g., 50, 40, 30, 20, 10, 9, 8,
7, 6, 5, 4, 3, 2, or fewer cells. For example, hydrogel
micro-carrier carries one single cell. For example, the hydrogel
micro-carrier surrounds a cell, e.g., a single cell, on one or more
(e.g., all) dimensions of the cell, e.g., the hydrogel
micro-carrier encapsulates the cell. In some cases, the hydrogel
micro-carrier is in the form of a liquid droplet, e.g., of
picoliter volume. For example, the droplet has a volume of about
0.1-1000 pL, e.g., about 1-1000 pL, 1-500 pL, 1-100 pL, 10-500 pL,
or 10-100 pL.
[0280] Hematopoietic factors are proteins that cause blood cells,
e.g., HSCs, to grow and/or mature. For example hematopoietic
factors regulate, e.g, enhance, blood production. Exemplary
hematopoietic factors include stem cell factor (SCF), interleukin-2
(IL-2), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin-7
(IL-7), granulocyte-macrophage colony stimulating factor (GM-CSF),
granulocyte colony stimulating factor (G-CSF), macrophage colony
stimulating factor (M-CSF), erythropoietin, thrombopoietin,
collagen-I, interleukin-11 (IL-11), angiopoietin-1, and
transforming growth factor-beta (TGF-beta).
[0281] In some examples, cell growth/behavior differs relative to
the disease state of a given tissue, e.g., the shear modulus (a
measure of stiffness) of normal mammary tissue is approximately 100
Pascal, whereas that of breast tumor tissue is approximately 2000
Pascal. Similarly, normal liver tissue has a shear modulus of
approximately 300 Pascal compared to fibrotic liver tissue, which
is characterized by a shear modulus of approximately 800 Pascal.
Growth, signal transduction, gene or protein expression/secretion,
as well as other physiologic parameters are altered in response to
contact with different substrate stiffness and evaluated in
response to contact with substrates characterized by mechanical
properties that simulate different tissue types or disease states.
In one embodiment, the stiffness of a hydrogel is tuned to enhance
hematopoiesis. For example, the stiffness of a hydrogel is tuned to
enhance secretion of hematopoietic factors from MSCs.
[0282] In accordance with any method described herein, a subject as
described herein is a mammal, e.g., a human, dog, cat, horse, cow,
pig, goat, sheep, rabbit, or monkey. In some examples, a subject
described herein is one who suffers or has suffered from a cancer,
immune deficiency disorder, or a blood disease. For example, the
cancer comprises a blood cancer or a solid tumor cancer. Exemplary
blood cancers include a leukemia, lymphoma, and myeloma. Solid
tumor cancers, e.g., comprise an adrenocortical tumor, colorectal
carcinoma, breast cancer, lung cancer, ovarian cancer, uterine
cancer, endometrial cancer, cervical cancer, gliobastoma, colon
cancer, stomach cancer, pancreatic cancer, desmoid tumor,
desmoplastic small round cell tumor, endocrine tumor, Ewing
sarcoma, hepatocellular carcinoma, melanoma, neuroblastoma,
osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms tumor,
nasopharyngeal cancer, testicular cancer, thyroid cancer, thymus
cancer, gallbladder cancer, central nervous system (CNS) cancer,
bladder cancer, or bile duct cancer. Exemplary blood diseases
include thalassemia, aplastic anemia, and sickle cell anemia. Also,
exemplary immune deficiency disorders include X-linked
agammaglobulinemia (XLA), severe combined immunodeficiency (SCID
disorder), common variable immunodeficiency, alymphocytosis.
[0283] In some cases, a subject as described herein has undergone
or is undergoing a chemotherapy or a radiation treatment.
[0284] In addition to hematopoietic factors, cells, e.g., MSCs,
also secrete other factors, e.g., ones that ameliorate myocardial
infarction. As such, the invention further provides a method for
treating or preventing a cardiovascular disease in a subject in
need thereof, comprising administering to the subject a hydrogel
described herein, e.g., a 2-dimensional or 3-dimensional hydrogel
comprising a stiffness and a mesenchymal stem cell (MSC). For
example, the cardiovascular disease comprises coronary artery
disease, cardiomyopathy, hypertensive heart disease, heart failure,
cor pulmonale, cardiac dysrhythmia, indocarditis, inflammatory
cardiomegaly, myocarditis, valvular heart disease, cerebrovascular
disease, peripheral arterial disease, congenital heart disease, or
rheumatic heart disease. For example, hydrogel micro-carriers that
contain MSCs are injected directly to a damaged heart. In some
examples, the hydrogel micro-carriers provide prolonged delivery of
factors that ameliorate myocardial infarction compared to direct
administration of the factors or administration of MSCs that are
not encapsulated in hydrogel micro-carriers.
[0285] Exemplary factors that promote cardiovascular regeneration,
e.g., that ameliorate myocardial infarction, include vascular
endothelial growth factor (VEGF), stromal cell derived factor 1
(SDF-1), tumor necrosis factor-inducible gene 6 protein (TSG-6),
interleukin-6 (IL-6), interleukin-8 (IL-8), basic fibroblast growth
factor (bFGF or FGF-2), insulin-like growth factor 1 (IGF-1),
hepatocyte growth factor (HGF), Thrombospondin-4, secreted
frizzled-related protein 2 (Sfrp2), matrix metalloproteinase 9
(MMP-9), tissue inhibitor of metalloproteinases (TIMP)
metallopeptidase inhibitor 2 (TIMP-2), monocyte chemotactic protein
1 (MCP-1), thrombospondin 1 (TSP-1), chemokine (C-X-C motif) ligand
6 (CXCL6), interferon gamma-induced protein 10 (IP-10). For
example, one of more of these factors are secreted by a MSC.
[0286] In some cases, the hydrogel comprises 50 or fewer (e.g., 50,
40, 30, 20, 10, or fewer) MSCs. For example, the hydrogel comprises
one single MSC.
[0287] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In the case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and are not intended to be
limiting.
[0288] The invention will be further illustrated in the following
non-limiting examples.
Examples
[0289] The following materials and methods were used in Examples
1-7.
[0290] Statistical Analysis. For the experiments described below,
two-tailed student t-test is performed with the data from .gtoreq.3
experiments. Multiple group analysis is done using ANOVA. P<0.05
is considered statistically significant between control and
perturbations.
[0291] Sources of cells and tissues. Adult human stem cells from
bone marrow are purchased from commercial vendors. Primary mouse
tissues are obtained using established mouse strains.
[0292] Species/Strain/Age/Sex
[0293] Mice, NOD/scid-IL-2Rgc null (NSG, JAX stock 005557). Animals
are normally studied between 2-6 months of age. Both sexes are used
in the experiments described herein.
[0294] Genomic DNA Testing
[0295] In some cases, development and breeding of genetically
modified animals are not performed with recombinant DNAs.
[0296] Peripheral Blood Sampling
[0297] For a period up to 5 months after the transplantation, the
mice are anesthetized using isoflurane inhalation, and a small
amount of blood (50 .mu.L per adult mouse, about 25-35 g) is
sampled from the transplanted mice from the retro-orbital capillary
bed once a month. The mice are euthanized afterwards.
[0298] Human Bone Marrow (BM) Cell Xenotransplantation
[0299] Human BM CD34+ hematopoietic stem cell and progenitors
(HSC/Ps), with or without culture, are transplanted either via the
intrabone or intravenous route into sublethally irradiated
(.about.250 rads) NSG mice, which is the optimal dosage to keep
mice alive in sterile conditions, while clearing enough host mouse
blood cells to maintain human donor cells in vivo. Recipient mice
are subjected to the transplantation within 8 hours of sublethal
irradiation. The animals are fed with 8 mL of antibiotic Septra
water for two weeks after cell transplantation. The animals are
observed 3 times a week for the duration of each experiment,
followed by euthanization. Any sick mice, which show signs of
dehydration, hunched posture, or decreased activity will be
euthanized. The total blood counts are typically recovered to
normal levels in less than two weeks.
[0300] Human Mesenchymal Stromal Cell (MSCs)
Xenotransplantation
Human MSCs are isolated by plastic adherence and expanded, followed
by incorporation into polymer constructs. Up to two cylindrical
polymer discs with or without MSCs (1 cm diameter, 0.5 cm height)
are implanted subcutaneously per NSG mouse by performing survival
surgery. Briefly, mice are anesthetized utilizing ketamine/xylazine
injection (80-120 mg/kg ketamine+5-10 mg/kg xylazine per mouse). A
small incision (1-3 cm) is then be made through the subcutaneous
tissue, and scissors are used to create a small pocket suitable for
the implantation. Several stitches (Maxon 7-0, a monofilament
synthetic absorbable suture) are placed within the subcutaneous
pocket to immobilize the polymer. The incisions are then closed
with closure clips (REFLEX' clips). The animals are allowed to
recover from anesthesia and returned to cages. Skin sutures or
clips are removed within 10-14 days post-surgery, and the longest
duration for implants is 6 months. Human BM CD34.sup.+
transplantation in these mice is done in limiting dilution 8 weeks
after MSC implantation to test human HSC engraftment in the newly
formed ectopic niche.
Anticipated Numbers of Animals
[0301] In the studies described herein, the number of animals
required per arm is estimated based on prior studies that would be
the minimum needed to reach a statistical difference of 0.05 by a
Student t-test.
Breeding
[0302] Animals are procured from the Jackson Laboratory which
provides both naive NOD-scid-IL-2Rgc null mice. Animals are bred
according to standard protocols. If there are 10 breeding pair per
year, 10 pair.times.2 mice/pair (male and female).times.2 years=40
mice for breeding purposes. For the studies involving the
quantification of human HSC number by limiting dilution from human
BM CD34.sup.+ cells cultured in the presence of human BM
MSC-conditioned medium, the total number of animals is calculated
as follows: 1. Different stiffness for human BM MSCs: soft (1 kPa),
intermediate (10 kPa), and stiff (40 kPa) cultured in either
undifferentiating or differentiating medium=3.times.2=6 medium
conditions; 2. Undifferentiating or differentiating medium without
MSCs=2 medium controls; 3. Different stiffness for human BM HSCs:
very soft (0.05 kPa) and soft (1 kPa)=2; 4. The number of limiting
dilution doses=3; 5. The number of mice per dose=5; with a total
number of animals of (6+2).times.2.times.3.times.5=240 mice. For
studies involving the subcutaneous implantation of human BM MSCs in
polymer scaffolds, followed by the ectopic niche formation and
subsequent transplantation with different doses of human BM
CD34.sup.+ cells to test HSC engraftment in the ectopic niche in
vivo, the total number of animals is calculated as follows: 1. Bulk
hydrogel with different stiffness for human BM MSCs: soft (1 kPa),
intermediate (10 kPa), and stiff (40 kPa)=3 conditions; 2. Single
encapsulated MSC droplets mixed in different ratios. soft (1
kPa):stiff (40 kPa)=1:9, 5:5, 9:1=3 conditions; 3. The number of
limiting dilution doses=3; 4. The number of mice per dose=5, 2
scaffolds per recipient; with a total number of animals of
(3+3).times.3.times.5=90 mice. Thus, the total number of animals
used for the studies described herein is about 40
(Breeding)+240+90=370 mice.
[0303] In some examples, an immune-deficient mouse is used to
characterize human hematopoietic and mesenchymal cells in vivo. For
example, immune-deficient mice represent the only in vivo model for
the study of human hematopoietic niche and blood cell formation and
for the development of therapeutic strategies for blood disorders.
This model is used for the study of bone formation, angiogenesis
via human endothelial progenitors in ischemic muscle injury models,
and human hematopoietic system reconstitution. Instead of NSG mice,
alternative previous generation immuno-deficient mice models,
including nude and NOG/SCID mice, can be used.
[0304] Mouse models are useful for developing mechanically
controlled strategies to form the BM niche and engineer blood
formation and for recapitulating the complexity of an animal blood
network. In some examples, in vivo studies described herein
demonstrate the functionality of HSCs to reconstitute blood
long-term, and to induce hematopoietic niche formation by MSCs
incorporated in designed polymer constructs. The results herein
show that gel/matrix/substrate mechanics play roles in HSC
differentiation in vitro. The therapeutic potential of mechanically
controlling the BM niche formation to engineer blood formation is
further confirmed, e.g., by studying mice xeno-transplanted with
human cells. For example, only immune-deficient mice will accept
human xenografts. These immune-deficient mice are sensitive to
pathogens--as such, mice are handled in a sterile environment,
e.g., in a BSL-2 space equipped with double HEPA-filtered
microisolator cages. Mice are given irradiated chow and acidified
water, and they are housed in microisolator cages contained in
ventilated racks. Personnel handling the mice wear gowns, caps,
masks, foot coverings and sterile gloves. All manipulations of the
mice such as feeding and changing cages are done under a laminar
flow hood within the sterile room.
[0305] Intrabone or intravenous injections of primary human
CD34.sup.+-derived cells are performed in mice. In some cases,
sublethal irradiation can lower the total blood count for at least
two weeks post-irradiation and transplantation. Injected animals
are monitored within 24 hours of the injection, and then at least 3
times a week thereafter. Prophylactic Septra water and wet food are
provided on the cage floor for all recipient mice for two weeks
post-transplantation. If the mice display a marked decreased
activity, decreased intake of water and food, and hunched posture,
they are euthanized. Mice experiencing weight loss greater than 30%
within a 14-day period are sacrificed as well. Euthanasia comprises
CO2 narcosis as approved by the Panel on Euthanasia of the American
Veterinary Medical Association, and e.g., animals are observed for
15 min afterwards for loss of heartbeat and movement.
[0306] Materials and experimental methods used in Examples 8-11 are
as follows.
Microfluidic Device and Microwell Fabrication
[0307] Soft lithography was used to fabricate microfluidic devices
and microwells. Negative photoresist SU-8 3025 (MicroChem, Newton,
Mass.) was deposited onto clean silica wafers to a thickness of 25,
50, 100 or 200 .mu.m, and patterned by UV light exposure through a
transparency photomask (CAD/Art Services, Bandon, Oreg.). After the
photoresist was developed, polydimethylsiloxane (PDMS) (Dow
Corning, Midland, Mich.) was mixed with crosslinker (ratio 10:1),
degassed, poured, and cured for at least 1 hour at 65.degree. C.
For microfluidic devices, the PDMS replicas were peeled off the
wafer and bonded to glass slides by oxygen-plasma activation of
both surfaces. Microfluidic channels were then treated with Aquapel
(PPG Industries, Pittsburgh, Pa.) by passing the solution through
the channels, to improve wetting of channels with fluorinated oil.
Polyethylene tubing with inner diameter 0.38 mm and outer diameter
1.09 mm and 27G.times.1/2 needles were used to connect channels to
plastic syringes (all from Becton Dickinson, Franklin Lakes,
N.J.).
[0308] PDMS microwell replicates were peeled off of the wafer, and
3D-printed polyurethane structures were glued onto the microwell
fields using PDMS. After curing for at least 1 hour at 65.degree.
C., microwell-structures were incubated in 70% ethanol for 2 h.
Microwell fields were washed with deionized H.sub.2O and treated
with 3% Pluronic F68 for 10 minutes under vacuum, followed by two
washes with Dulbecco's phosphate buffered solution. Hydrogel
capsule-encapsulated cells suspended in complete DMEM were then
seeded and allowed to settle by gravitational sedimentation for 1
h.
Alginate Preparation and Formation of Bulk Hydrogels
[0309] Sodium alginate with high molecular weight and high
guluronic acid content was purchased from FMC Biopolymer
(Princeton, N.J.). To produce lower molecular weights alginate, the
high molecular weight alginate was irradiated by a 3 Mrad cobalt
source. Alginates were covalently coupled with the integrin-binding
peptide (Gly)4-Arg-Gly-Ala-Ser-Ser-Lys-Tyr (SEQ ID NO: 13)
(Peptides International) and either Fluoresceinamine, isomer I
(Sigma-Aldrich) or Lissamine.TM. Rhodamine B Ethylenediamine
(Setareh Biotech).
[0310] Bulk alginate hydrogels were fabricated with a calcium
sulfate slurry as the calcium source. Alginate was transferred to a
3 mL syringe, and the calcium slurry to another syringe (BD). The
two syringes were connected with a female-female Luer lock coupler
(ValuePlastics), without introducing air bubbles, and the two
solutions were rapidly together with eight pumps of the syringe
handles. The alginate was deposited on a glass plate and allowed to
cross-link for 45 minutes. Bulk alginate gels were either 1%
alginate for lower elastic moduli, or 2% for higher elastic
moduli.
Cell Culture
[0311] Clonally derived mouse MSCs (D1s) purchased from American
Type Cell Culture (ATCC) were expanded subconfluently in
high-glucose, 10% fetal bovine serum-supplemented Dulbecco's
Modified Eagle media (complete DMEM). OP9s were purchased from ATCC
and expanded subconfluently in Alpha Minimum Essential Medium
supplemented with 20% fetal bovine serum. OECs were isolated from
human cord blood within 12 hours from collection using standard
techniques. Colonies were replated and expanded subconfluently in
EGM-2MV (Lonza #CC-3202, Walkersville, Md.) media prior to use, and
cells were used for studies between passages 5-7.
Encapsulation
[0312] Calcium carbonate nanoparticles (CalEssence.RTM. 70 PCC)
were suspended in complete DMEM buffered with pH 7.7 25 mM HEPES
(HEPES-DMEM) (Sigma-Aldrich, St. Louis, Mo.) and sonicated for 15
seconds with a Vibra-Cell Sonicator at 70% amplitude. The
suspension was mixed with 25 mL HEPES-DMEM, centrifuged for 50 rcf
for 5 minutes, and the supernatant removed. This was spun at 1000
rcf, and the pellet was resuspended to 10 mg/mL, based on initial
mass. Cells were incubated in nanoparticle suspension for 40
minutes with gentle agitation. Excess nanoparticles were removed
using centrifugation, and the coated cells were suspended in
complete DMEM buffered with 50 mM HEPES to pH 7.7. This was
combined with polymer precursor solutions for injection. In direct
injection experiments, cells at the same concentration as in the
pre-coated experiment were directly mixed with calcium carbonate
nanoparticles before combining with alginate. The continuous phase
was prepared by mixing 1% fluorosurfactant (Holtze 2008) and
sterile-filtered 0.31% acetic acid (EMD Chemicals, Gibbstown, N.J.)
in a fluorinated oil (3M.TM. Novec.TM. Engineering Fluid HFE-7500).
The continuous and aqueous phases were injected into the
microfluidic device at flow rates of 3.2 uL/min and 1 uL/min,
respectively. Emulsions were broken after a 40 minute incubation by
the addition of 20% 1H,1H,2H,2H-perfluorooctanol (Alfa Aesar).
[0313] To fabricate hybrid collagen-alginate hydrogel capsules, a
cell and polymer precursor suspension containing 3 mg/mL calcium
carbonate nanoparticles, 0.93% 139 kDa alginate, and 0.66 mg/mL rat
tail collagen I (Corning, Bedford, Mass.) was injected at 4.degree.
C. into a microfluidic device of the same design as that used for
alginate hydrogel capsules and operated with the same parameters.
The resulting emulsion was incubated at 37.degree. C. for 30
minutes. To fabricate hybrid fibrin-alginate hydrogel capsules, two
solutions were prepared: one combining fibrinogen (20.3 mg/mL) and
aprotinin (45 ug/mL); and one combining calcium carbonate
nanoparticles (6.7 mg/mL), 2.1% alginate, and thrombin (22 U/mL).
Cells were suspended in the solution containing fibrinogen. The two
aqueous phases and the continuous phase were injected into separate
inlets of the microfluidic device at flow rate of 0.5 uL/min and
3.2 uL/min, respectively.
Analysis of Cell Egress and Hydrogel Capsule Size
[0314] Alginate hydrogel capsules encapsulating cells were
themselves encapsulated in a bulk collagen hydrogel. Following
manufacturer's instructions, rat tail collagen I (Santa Cruz
Biotechnology, Santa Cruz, Calif.) was first mixed with Dulbecco's
phosphate buffered saline and sodium hydroxide to achieve a neutral
pH, and then mixed with a suspension containing cells encapsulated
in alginate hydrogel capsules to obtain a final collagen
concentration of 1.85 mg/mL. The suspension was added to wells in a
48-well plate and allowed to cross-link at 37.degree. C. for 30
minutes. Collagen gels were fixed after 1 day of culture. Cells
were stained with rhodamine- or fluorescein-conjugated phalloidin
(Biotium, Hayward, Calif.) and DAPI, and imaged with a Nikon E800
upright microscope. Only hydrogel capsules that showed a morphology
of having contained cells (e.g., hollowed out) were considered to
have led to cell-egress.
Mechanical Testing
[0315] Prior to atomic force microscopy measurement, encapsulated
cells in fluorescently labeled alginate were adhered to a
polylysine-coated glass slide. Glass microscope slides (VWR
International, Radnor, Pa.) were cleaned in a solution of 10%
sodium hydroxide and 60% ethanol, rinsed with deionized water, and
incubated with poly-L-lysine (Handary S A, Belgium). MFP-3D system
(Asylum Research) was used to perform AFM measurements of Young's
modulus of hydrogels, using silicon nitride cantilevers (MLCT,
Bruker AFM Probes). The stiffness was calibrated by determining a
spring constant of the cantilever from the thermal fluctuations at
room temperature, ranging from 20.about.50 mN/m. The cantilever was
moved towards the stage at a rate of 1 .mu.m s.sup.-1 for
indentations. For bulk hydrogels, a disc of 5 mm.times.2 mm was
placed onto a PDMS mold on a glass slide. Force-indentation curves
were fit using the Hertzian model with a pyramid indenter. The
elastic modulus of bulk alginate hydrogels was measured by casting
10-mm diameter and 2-mm thick cylindrical discs and compressing
without confinement using an Instron 3342 mechanical apparatus at 1
mm min.sup.-1.
Osteogenic Induction and Analysis of Alkaline Phosphatase (ALP)
Production
[0316] To induce osteogenesis, D1s encapsulated in hydrogel
capsules were cultured with complete DMEM supplemented with 10 mM
.beta.-glycerophosphate and 250 .mu.M L-ascorbic acid, cycling
every two days. D1s were fixed six days after osteogenic induction
and stained with elf-97, following the manufacturer's instructions.
Staining was stopped through washing with excess of PBS after 90
seconds. Fixed cells were further stained with rhodamine-conjugated
phalloidin (Biotium, Hayward, Calif.). Fluorescence images for
immunohistochemistry, elf-97 staining, and alginate were acquired
using an Olympus IX81 inverted microscope equipped with a Cary II
Nipkow-type Spinning Disc Confocal Attachment (BD Biosciences, San
Jose, Calif.) and a Coolsnap HQ2 camera (Prior Scientific,
Rockland, Mass.). The area-average fluorescence of cells stained
with elf-97 and of alginate was quantified with ImageJ (FIG.
21).
Expression of Exogenous Genes in Cells
[0317] To introduce mCherry and Firefly Luciferase in MSCs,
lentiviral particles containing the vector with
mCherry-IRES-Firefly Luciferase driven by the CMV promoter were
purchased from the Vector Core at Massachusetts General Hospital.
Cells were incubated with viral particles for 2 days. Cells
expressing mCherry were then sorted via flow-activated cell sorting
(FACS). In some cases, Cyan Fluorescence Protein (CFP) and Gaussia
Luciferase were introduced to MSCs using the same approach.
Animal Experiments
[0318] All animal experiments were performed in accordance with
institutional guidelines approved by the ethical committee from
Harvard University. To evaluate the biodistribution of donor cells
in vivo, MSCs expressing firefly luciferase either with or without
single cell encapsulation were injected into mice. Subsequently, 3
mg of D-luciferin was injected intraperitoneally into the 25 g mice
followed by luminescence imaging with the IVIS Spectrum
(PerkinElmer) at indicated times. The systemic secretions of donor
cells were evaluated in two ways. For allogeneic transplantation,
mMSCs from Balb/c mice expressing Gaussia luciferase were injected
into C57/BL6 mice, followed by blood collection at regular time
intervals. 10 uL blood was mixed with 100 uL of 20 ug/ml
coelenterazine-h substrate in a white, opaque 96-well plate and
luminescence was detected using a BioTek microplate reader. For
xenograft, human MSCs were injected into NOD/SCID/IL-2.gamma.-/-
mice. For each sample, 50 .mu.L of blood plasma was used to
evaluate the systemic level of human IL-6 by using an ELISA kit
(R&D Systems).
Example 1: Alginate Hydrogel Recapitulated a Range of Mechanical
Properties of the Native Marrow
[0319] To model a range of bone marrow (BM) mechanics in vitro, an
alginate-based hydrogel conjugated with an integrin-binding RGD
peptide was used. Alginate is non-adhesive to cells, so the
adhesion to gels was mostly through the RGD. Mechanical properties
of the hydrogel were controlled by Ca.sup.2+ cross-linking and gel
concentrations (FIG. 2A). Without gel cross-linking, 1% of the low
molecular weight alginate ("10/60", 150 kDa) fluid recapitulated
the known viscosity value of BM (40.about.400 cP) (Gurkan and
Akkus, 2008, Ann Biomed Eng 36, 1978-1991). The minimum [Ca.sup.2+]
required for gelation of the 1% 10/60 alginate was 10 mM with E=50
Pa (FIG. 2B). In contrast, the same [Ca.sup.2+] led to E=1000 Pa
with 1% of the higher molecular weight ("20/40", 250 kDa) alginate
or 2% of 10/60 alginate. Since increasing [Ca.sup.2+] beyond 10 mM
may affect cell viability, a combination of RGD-modified and
unmodified alginate gels were used to further increase stiffness by
increasing gel concentration but maintaining the same RGD
density.
Example 2: Matrix Stiffness Regulated the Release of Paracrine
Factors from MSCs that Control the Number of an HSC-Enriched
Subpopulation In Vitro
[0320] Matrix stiffness directly regulates HSC/P number in 2D
culture with tropoelastin matrix (Hoist et al., 2010, Nat
Biotechnol 28, 1123-1128). Here, matrix stiffness monotonically
decreased the number of a human HSC-enriched subpopulation (defined
as CD34+CD38-CD133+CD90+) (Majeti et al., 2007, Cell Stem Cell 1,
635-645) in the 3D alginate hydrogel with the half-maximal
value=1.3 kPa (FIG. 3A). While the HSC-enriched population was
highly sensitive to matrix stiffening, MSCs showed distinct
biological responses, such as cell spreading and differentiation,
across a large dynamic range of matrix stiffnesses (0.1-100 kPa)
(Engler et al., 2006, Cell 126, 677-689; Huebsch et al., 2010, Nat
Mater 9, 518-526). The ability of matrix stiffness to further
regulate HSC/P differentiation by modulating paracrine secretion
from MSCs was examined by using a transwell assay with 400 nm pore
polycarbonate membrane to physically separate HSC/Ps and MSCs.
Human BM CD34+ cells were encapsulated in the 3 kPa gel in the
lower chamber, and MSCs were put in gels with a range of stiffness
(1-30 kPa) in the upper chamber. Soluble factors from MSCs at 10
kPa further increased the HSC-enriched subpopulation number (FIG.
3B), indicating that an optimal stiffness exists for MSCs to
release factors that maximize the HSC number.
Example 3: Encapsulation of Single Cells in Alginate Hydrogel with
Distinct Matrix Stiffness
[0321] A droplet microfludics-based method (Guo et al., 2012, Lab
Chip 12, 2146-2155, incorporated herein by reference) was adapted
to encapsulate a few cells or single cells in alginate-based gel
droplets (FIG. 4A), which were then immobilized in a micro-well to
characterize their mechanical properties by atomic force microscopy
(AFM) (FIG. 4B). The analysis showed that it was possible to make
alginate droplets with biologically relevant stiffness by changing
initial calcium cross-linker concentrations (FIG. 4C) or alginate
concentrations.
Example 4: Systematically Investigate and Validate Experimental
Conditions where HSC-Enriched Population can be Maximized
[0322] Experiments are conducted to elucidate mechanisms behind
matrix stiffness-dependent release of hematopoietic factors from
MSCs, e.g., to determine how matrix stiffness regulates paracrine
release of hematopoietic factors from MSCs. (FIG. 5).
[0323] The results described above showed that HSC enrichment can
be maximized by putting together CD34.sup.+ cells in soft gels and
MSCs in intermediate stiffness gels via a transwell setting (FIG.
3). Here, additional experimental conditions are tested for their
ability to maximize HSC-enriched population. Homogenous populations
of human MSCs (CD146.sup.+) as defined previously (Sacchetti et
al., 2007, Cell 131, 324-336) are tested. Also, different ratios
between MSCs and HSCs are tested. For example, HSCs are
encapsulated in soft (<100 Pa) gels to ensure the maximum
HSC-enriched number. To validate the findings with human cells,
mouse cells (D1 MSCs and
Lin.sup.-Sca-1.sup.+cKit+CD48.sup.-CD150.sup.+HSC/Ps) are tested.
To test the generality of the relationship between matrix
elasticity and paracrine regulation of hematopoiesis by MSCs,
RGD-modified agarose or poly(ethylene glycol dimethacrylate)
hydrogels of varying rigidity are used, as previously described
(Huebsch et al., 2010, Nat Mater 9, 518-526).
Example 5: Determination of the Roles of Extracellular Vesicles
Released from MSCs on HSC/P Differentiation
[0324] Since BM MSCs are known to secrete exosomes and impact blood
cancer cell proliferation (Roccaro et al., 2013, J Clin Invest 123,
1542-1555), the present study initially focuses on this mode of
secretion to assess its impact on normal hematopoiesis. Since the
alginate gels used in this study have a pore size of <100 nm and
the transwell membrane filter has that of .about.400 nm, it is
likely that the paracrine effects described in FIG. 3B are due to
either exosomes or soluble factors, but not microvesicles (.about.1
.mu.m). The experiments described in this example focus on
quantifying and characterizing potential hematopoietic factors
released from MSCs cultured in different matrix rigidity. In some
cases, to minimize the contribution from initial cell-cell contact
on the MSC secretome, cells are cultured at low density (e.g., 1
million cells/mL or lower, e.g., 10.sup.6, 5.times.10.sup.5,
1.times.10.sup.5, 0.5.times.10.sup.5 cells/mL or lower) by
controlling cell number to gel volume ratios.
[0325] In some cases, if exosomes or soluble factors released from
MSCs in standard alginate gels with pore size <100 nm (FIG. 2)
do not play significant roles in hematopoiesis, macroporous
cryogels with larger pore size (>10 .mu.m) (Bencherif et al.,
2012, Proc Natl Acad Sci USA 109, 19590-19595) will be tested to
address if larger particles such as microvesicles from MSCs in
different stiffness impact hematopoiesis.
[0326] Isolation and quantification of MSC exosome release.
Exosomes are isolated from the conditioned media of MSCs cultured
in alginate hydrogels with various stiffnesses, using size
exclusion filters and ultracentrifugation as described previously
(Roccaro et al., 2013, J Clin Invest 123, 1542-1555). The isolated
exosomal content is then quantified by performing Western blotting
with antibodies against exosome-specific markers (e.g. CD63 and
CD81) and normalizing the signal by the total protein content for
each sample. Alternatively, electron microscopy is used to count
the number of exosomes labeled by their markers with a standard
immunogold method. By counting the number of exosomes from MSCs at
different culture time points, it is possible to calculate the
general exosome release kinetics and model it as a function of
matrix rigidity.
[0327] Effects of MSC exosomes on HSC/Ps ex vivo. An equal amount
of exosomes isolated from MSCs at day 1, 3, and 7 cultures in
different stiffnesses are directly applied to human BM CD34.sup.+
cells encapsulated in the soft gel (.about.100 Pa). After a 7-day
culture, flow cytometry is used to evaluate HSC/P subpopulations
and hematopoietic lineages as described in FIG. 3. If significant
expansion of HSC-enriched population is observed, HSC number will
be functionally quantified by collecting cultured
CD34.sup.+-derived cells, serially diluting and xenotransplanting
in sub-lethally irradiated (250 cGy)
NOD/Shi-scid/IL-2R.gamma..sup.-/- (NSG) mice. Human reconstitution
in the recipient blood is analyzed at 4, 8, and 16 weeks after
transplantation using an antibody against human-CD45 and CD47. Mice
are then sacrificed to analyze BM engraftment. To functionally
assess human hematopoietic progenitors, cells from culture are
plated into semi-solid methylcellulose medium with growth factors
to quantify colony-forming unit (CFU)--granulocyte-macrophage (GM),
CFU--granulocyte, erythroid, macrophage, megakaryocyte (GEMM), and
burst-forming unit (BFU-E). In some cases, instead of
xenotransplantation to quantify human HSC number, studies are done
with mouse BM D1 MSCs and HSC/Ps from donor mice (CD45.2), followed
by competitive transplantation of donor cells into irradiated
CD45.1 recipient mice.
[0328] Hematopoietic factors released from MSCs. To address the
possibility that the content of secreted hematopoietic proteins
from MSCs is changed by matrix elasticity, isolated exosomes, or
more broadly, conditioned media concentrated with the 3 kDa cut-off
filter is subjected to separation by SDS-PAGE gel electrophoresis.
The gel is stained with Coomassie blue and selected bands (<50
kDa) are excised. Sequencing grade-modified trypsin is used to
digest proteins in the bands. Peptides are separated by liquid
chromatography, followed by mass spectrometry through a
LTQ-Orbitrap XL ("LC-MS/MS"). Raw mass spectrometry data is
annotated by SEQUEST (>3 peptides per protein). The label-free
"Peptide Ration Fingerprint" algorithm is used to quantify peptides
in different samples (Swift et al., 2013, Science 341, 1240104). In
some cases, instead of LC-MS/MS to analyze proteomes,
antibody-based protein array methods will be used (Parekkadan et
al., 2007, PLoS One 2, e941).
Example 6: Mechanisms Behind Mechanical Regulation of Factor
Release from MSCs
[0329] To confirm the immediate involvement of actomyosin forces in
mechano-sensitive factor release, MSCs cultured in hydrogels with
different matrix stiffnesses for 1-2 days are treated with
blebbistatin (myosin-II ATPase inhibitor) or Y-27632
(Rho-associated protein kinase inhibitor) for 1 day, and their
exosome numbers are quantified. For indirect long-term regulation,
mechanical stress triggered by stiff substrates activates
mechano-sensitive transcription factors and alters gene expression
to further augment cellular tension (Wang et al., 2009, Nat Rev Mol
Cell Biol 10, 7582), which may then affect exocytosis. Therefore,
whether or not the mechano-sensitive factor release from MSCs also
requires transcription factors is tested (FIG. 5). Transcription
factors are activated by matrix stiffness either indirectly by
biochemical changes in cytoskeletal networks or directly by
attachment to the force-generating cytoskeleton (Janmey et al.,
2013, Differentiation 86, 112-120). Representative transcription
factors from indirect and direct modes of activation are tested:
Megakaryocyte acute leukemia protein (MAL)/Serum Response Factor
(SRF) (Miralles et al., 2003, Cell 113, 329-342) and Yes-associated
protein (YAP)/Transcriptional coactivator with PDZ-binding motif
(TAZ) (Dupont et al., 2011, Nature 474, 179-183), respectively.
Short hairpin RNA (shRNA) lentivirus is used to downregulate these
transcription factors in MSCs, followed by testing their ability to
regulate HSC/P differentiation using a transwell assay across
different stiffnesses. Both exosome release kinetics and soluble
factor contents are also evaluated after shRNA downregulation.
Example 7: Infusible Hydrogel Micro-Carriers that can Mechanically
Trigger Hematopoietic Factor Release from Encapsulated MSCs In
Vivo
[0330] Bulk hydrogels have been used for subcutaneous implantation
of cells, but they are not currently suitable for other routes of
in vivo administration, including intravenous or intrabone. MSCs in
hydrogels have been implanted subcutaneously to study their
indirect impact on hematopoiesis by forming a bone nodule, which
will then recruit marrow cells. To study the direct impact of MSCs
on BM-resident HSC/Ps in vivo via hematopoietic factor release
triggered by substrate stiffness, a microfluidic method is
optimized to encapsulate individual MSCs in alginate gels, followed
by infusion for sustained delivery of secreted factors in vivo
(FIG. 6).
[0331] A microfluidic system to encapsulate single cells in
spherical alginate picoliter droplets was developed (FIG. 4). This
technique is adapted and used to vary matrix stiffness and size of
the droplets, place them in microwells, and confirm their
mechanical properties by AFM. In this way, single cell
encapsulation into hydrogels with controlled matrix stiffness and
size is achieved.
[0332] Single cell encapsulation: Cells, e.g., MSCs, are incubated
with CaCO.sub.3. After washing out excess CaCO.sub.3, the aqueous
phase (containing liquid RGD-conjugated alginate and cells) is
injected into a microfluidic system. A continuous flow of oil with
acetic acid is applied to pinch off the aqueous phase into beads
and dissolve CaCO.sub.3 into Ca.sup.2+ ions, which cross-link
alginate beads that encapsulate cells. To control substrate
rigidity, different concentrations of alginate or CaCO.sub.3 are
used. To control alginate bead diameters, a microfluidic channel
with different widths is used.
[0333] Microwell fabrication: Soft lithography is used to fabricate
microwells in poly(dimethyl siloxane) (PDMS) (Qin et al., 2010, Nat
Protoc 5, 491-502). A photomask is designed using computer-aided
design software and printed. The master is fabricated by shining
ultraviolet light on a silicon wafer with photoresist through the
photomask. PDMS is then polymerized using the master as a mold to
create microwells. Encapsulated cells are then seeded in each
well.
[0334] Mechanical testing of droplets by AFM: Single droplets in
PDMS microwells are probed with AFM. Young's modulus of each
droplet is calculated from linear indentation measurements with
pyramidal tip attached to a cantilever (Engler et al., 2007,
Methods Cell Biol 83, 521-545).
[0335] In vivo biodistribution of hydrogel droplets with different
substrate rigidity and size. The biodistribution of micro-scale
hydrogel particles is dictated both by their sizes and elastic
moduli, which likely affect circulation times upon intravenous
injection. In past studies, gel particles with lower elastic moduli
had higher circulation times (Merkel et al., 2011, Proc Natl Acad
Sci USA 108, 586591), but increasing particle diameters decreased
the circulation times (Kohane, 2007, Biotechnol Bioeng 96,
203-209). To control for the biodistribution kinetics while
altering substrate rigidity, alginate droplets are systematically
characterized in terms of elastic moduli (1-100 kPa) and diameters
(10-50 .mu.m) for their biodistribution kinetics in vivo. To
facilitate in vivo non-invasive imaging in living mice by the
Xenogen IVIS Imaging System (PerkinElmer), RGD-modified alginate is
conjugated with amine-cy5.5 (Lumiprobe) (excitation: 678 nm,
emission: 701 nm) to minimize background fluorescence from tissues.
The equal weight of alginate droplets is injected per mouse via
either an intravenous or intrabone route. For the intravenous
route, 200 .mu.l of 0.4 mg polymer/20 g mouse is injected
retro-orbitally (e.g. 10 million 20 .mu.m diameter 1% alginate
droplets). After injection, the IVIS is used to study particle
distribution across different organs. In addition, peripheral blood
is sampled and the presence of gel droplets in circulation is
detected by flow cytometry. Mice are analyzed every 30 min for the
first hour, followed by every two days for at least one week.
Droplets from blood samples are also sorted after 1-3 days of
infusion, and their integrities and mechanical properties are
confirmed by AFM. Post-mortem histological analyses are performed
on tissues, including lung, spleen, and marrow, to further
characterize the tissue localization of injected gel droplets.
[0336] Impact of MSCs encapsulated in hydrogel droplets with
different substrate stiffness on in vivo hematopoiesis. After
establishing appropriate size parameters to vary substrate
stiffness of hydrogel droplets without substantially altering their
in vivo biodistribution kinetics, human MSCs are encapsulated in
the droplets. Prior to infusion of MSC-droplets, human BM
CD34.sup.+ cells are intravenously transplanted into sub-lethally
irradiated NSG mice. MSC-droplets with varied stiffness are then
infused within 1 week or after 3 months of CD34.sup.+
transplantation to assess their effects on early HSC/P engraftment
and steady-state hematopoiesis, respectively. This will follow the
analysis of human blood reconstitution using flow cytometry. If
blood reconstitution is substantially enhanced by MSC-droplets, the
mice will be sacrificed to analyze the human HSC/Ps in BM by flow
cytometry. To functionally confirm whether enhanced human
hematopoiesis is due to HSC expansion, BM is serially transplanted
to new recipients followed by tracking human reconstitution up to 4
months. The efficacy of the MSC-droplets on hematopoiesis is
compared with the direct injection of soluble factors,
extracellular vesicles from MSCs, or non-encapsulated MSCs.
[0337] MSC homing after intravenous injection is a rare event
(Ranganath et al., 2012, Cell Stem Cell 10, 244-258), and in some
examples, the hydrogel further prevents homing. The presence of
non-hematopoietic human cells in recipient tissues is evaluated by
histological analysis to confirm whether any effect on
hematopoiesis by MSCs is due to their secreted factors. If
intravenous infusion does not lead to any satisfactory effect,
intrabone infusion of MSC-droplets will be performed and their
effects on hematopoiesis analyzed within a week to avoid effects
from MSCs integrating into BM. Although purifying alginate
increases biocompatibility, it is possible that alginate droplets
are physically trapped into spleen or lungs. While such an
entrapment will still allow factors to be secreted from MSCs (Lee
et al., 2009, Cell Stem Cell 5, 54-63), entrapment may increase the
probability for MSC-droplets to be engulfed by macrophages. If
significant clearance by macrophages occurs, alginate will be
conjugated with a minimal `self` peptide derived from CD47 that
impedes phagocytosis (Rodriguez et al., 2013, Science 339,
971-975). If ionic cross-linking of alginate hydrogels is too weak
for in vivo circulation, a bioorthogonal `click` chemistry method
will be used to covalently cross-link alginate. If size is not
sufficient to normalize the biodistribution kinetics caused by
changes in substrate stiffness, other parameters will be varied,
such as surface charge, e.g., by coating with poly-L-lysine.
Example 8: Encapsulation of Cells
[0338] D1 cells were first incubated with calcium carbonate
nanoparticles, which adsorbed to the cell surface (FIG. 16A) as a
function of nanoparticle concentration (FIG. 21A). After
non-adsorbed nanoparticles were washed away, coated cells were
mixed with liquid sodium alginate (FIG. 16B) and injected into a
microfluidic device (FIGS. 16C and 21B). Droplets of alginate, both
with and without cells, were pinched off by a continuous phase
composed of hydrofluoroether 7500, 1% fluorosurfactant, and 0.31%
acetic acid. Diffusion of the acid into the alginate droplet caused
a pH drop from 7.5 to 6.1 that, in the presence of nanoparticles,
was transient and mediated the dissolution of calcium from the
nanoparticles (FIG. 21C). Only cell- and nanoparticle-containing
droplets cross-linked. After a 30 minute incubation, the emulsion
was broken to retrieve the encapsulated cells (FIG. 16C).
[0339] Direct injection of a suspension containing cells, alginate,
and nanoparticles (not pre-adsorbed to cells) produced both empty
and cell-containing hydrogel capsules (FIG. 17A). When cells were
pre-coated with nanoparticles, encapsulation yield was
qualitatively higher (FIG. 17B). The alginate hydrogel formed a
thin layer around the encapsulated cells, ranging from 0.80 to 12
.mu.m and averaging 4.8 .mu.m thick (FIG. 17C-D). Moreover, both
the alginate within the hydrogel capsule (FIG. 17E), as assessed by
image analysis of confocal slices, and the population of hydrogel
capsules (FIG. 17F), as assessed by flow cytometry, followed a
unimodal distribution. Using a combination of flow cytometry and
image analysis, the coefficient of variation (CV) of the
fluorescent intensity of hydrogel capsules was 31%, and the CV of
hydrogel capsule size was 6.5%. In comparison, the polymer content
of empty hydrogel capsules had a CV of 14% and the hydrogel capsule
diameter had a CV of 3.4%. Because prolonged exposure to the oil
and surfactant phase decreased cell viability, acetic acid was
mixed into the oil and surfactant phase prior to injection to
immediately cross-link the alginate hydrogel capsule (FIG.
21D).
[0340] During the encapsulation process, not all of the
nanoparticles dissolved. A comparison between the amount of calcium
carbonate nanoparticles adsorbed to cell surfaces and the amount
dissolved from alginate and acetic acid (FIGS. 21A, C) showed that
the entire mass of nanoparticles did not dissolve during the
encapsulation process. To assess the impact of the remaining
nanoparticles on cells, the intracellular calcium levels of
encapsulated cells, non-encapsulated cells, and cells directly
exposed to nanoparticles was compared. No significant differences
in intracellular calcium levels were found between encapsulated and
non-encapsulated cells, though cells directly exposed to
nanoparticles exhibited higher intracellular calcium levels than
cells alone (FIG. 21E). The non-dissolved nanoparticles had no
impact on intracellular calcium levels in encapsulated cells (FIG.
21D).
[0341] OP9 cells, OEC cells, and D1 murine MSCs cells were
encapsulated (FIGS. 21-F-G). The number of cells per hydrogel
capsule for the three cell types was consistent, with 71%-81% of
the hydrogel capsules containing only one cell (FIG. 17G).
Encapsulation efficiency with pre-coated cells ranged from 74% for
OEC cells to 95% for OP9 cells and D1 murine MSCs (FIG. 17H). For
all cell types, the encapsulated yield of pre-coated cells was an
order of magnitude higher than of cells directly encapsulated
without pre-coating with nanoparticles (FIG. 17I). The expected
yields for direct injection into a microfluidic device (without
pre-coating cells with nanoparticles) were calculated from hydrogel
capsule size and starting cell density, with the assumption that
all alginate droplets would cross-link and that only one cell would
be contained in each cell-containing droplet. These expected values
were similar to experimental results for direct injection into the
microfluidic device (without pre-coating cells with nanoparticles)
(FIG. 17I). Yields for pre-coated D1s and OP9 were comparable to
the yield following flow cytometry sorting of D1 cells encapsulated
directly without nanoparticle pre-coating (FIG. 17I). The
encapsulation yield of OECs was poorer than that of D1 and OP9
cells, but still an order of magnitude higher with pre-coating of
nanoparticles than with direct encapsulation (FIG. 17I).
[0342] To assess the ability of small hydrogel capsules to support
cell viability in culture, encapsulated cells were stained with
calcein and ethidium homodimer one day and three days after
encapsulation (FIG. 17G). D1 and OP9 cells exhibited high viability
both one and three days after encapsulation. The viability of OECs
1 hour after encapsulation was high (0.77+/-0.22); at later time
points, their viability was lower. The encapsulation process was
unlikely to be responsible for this lower viability observed at
later time points. To further assess the ability of alginate gels
to support OECs, OECs were encapsulated in 8-mm.times.1-mm alginate
bulk hydrogels with calcium sulfate crosslinking and cultured; this
is a standard method for encapsulation of cells in alginate gels.
There were no significant differences in viability between OECs in
hydrogel capsules and bulk hydrogels, showing that it was likely
not the encapsulation step that led to the lower long-term
viability of OECs in the alginate gels.
Example 9: Alteration of Alginate Molecular Weight and
Mechanics
[0343] The egress of encapsulated cells as a function of polymer
molecular weight was analyzed. In the experiments described in
Example 8, D1s were encapsulated using polymer with a weight
average molecular weight of 139 kDa. In this example, higher (232
kDa) and lower (54 kDa) molecular weight alginates were also used
for cell encapsulation.
[0344] Cells encapsulated in different molecular weight alginates
were imaged (FIGS. 18A-C; 22A-C). The efficiencies of encapsulation
(fraction of cells encapsulated) did not vary significantly among
different molecular weights of alginate (FIG. 18D). The
encapsulation yield (fraction of hydrogel capsule beads containing
cells) of the 232 kDa alginate was significantly lower than that of
lower MW polymers (FIG. 18E). This difference is likely due to
increased shear from the higher viscosity of high MW alginate. For
all MWs, the encapsulation yield was much higher than that obtained
by direct injection into a microfluidic device without pre-coating
of nanoparticles. The long term viabilities of encapsulated D1
cells showed no significant differences among the three MW polymers
(FIG. 18F). The initial hydrogel capsule sizes were found to
exhibit slight, but significant differences, depending on polymer
MW (FIG. 18G).
[0345] Cells in hydrogel capsules formed from varying polymer
molecular weight were then encapsulated in a 11 mm.times.1.1 mm
collagen gel and cultured for one or three days. The diameter and
the number of cells per cell-containing hydrogel capsule were
quantified after three days of culture. Significantly fewer cells
per hydrogel capsule were found after three days of culture in 232
kDa alginate as compared to the lower MW polymers (FIGS. 18H and
22A-C). This difference in cell number was reflected in hydrogel
capsule size, as hydrogel capsules formed from 54 kDa and 139 kDa
alginate appeared to have increased in size over this time more
than hydrogel capsules formed from the 232 kDa polymer (FIG. 18I).
Additionally, after one day of culture, the number of empty
alginate hydrogel capsules and the number of hydrogel capsules that
still contained cells were counted to determine the fraction of
cells that had exited hydrogel capsules (FIG. 18J). The fraction of
cells that egressed was significantly higher in 139 kDa and 54 kDa
alginate beads than in 232 kDa alginate, showing that alginate MW
can be used to control cell trafficking out of hydrogel capsules
(FIG. 18K).
[0346] As extracellular matrix mechanics have been shown to
potently influence cell behavior, the ability of hydrogel capsules
to be further crosslinked post-fabrication was also analyzed. D1s
were encapsulated in 139 kDa and 232 kDa alginate hydrogel capsules
and subsequently exposed to 2 mM or 15 mM calcium chloride. The
elastic moduli of the hydrogel capsules were then measured using
atomic force microscopy (AFM). In hydrogel capsules of both
molecular weights, addition of calcium post-fabrication led to
increased elastic moduli (FIG. 18L).
[0347] To compare nanoscale measurements of elastic modulus with
those obtained at a macroscopic scale, 10 mm.times.2 mm alginate
hydrogels were fabricated, and their elastic moduli were measured
both with AFM and with an Instron mechanical apparatus (FIG.
22D).
Example 10: Encapsulating Cells in Other Polymers
[0348] As hybrid hydrogels have been shown to potentially combine
beneficial properties of each of their components, the ability to
form collagen-alginate and fibrin-alginate hybrid hydrogel capsules
with the methods described herein was tested. As cross-linking of
the secondary hydrogel (either collagen or fibrin) was not
dependent on the presence of calcium, cells were directly
encapsulated in hybrid hydrogel capsules without the nanoparticle
pre-coating step. Cell-encapsulating collagen-alginate hybrid
hydrogel capsules were fabricated by mixing calcium carbonate
nanoparticles, D1 cells, alginate, and neutral-pH liquid collagen
before injection into the microfluidic device at 4.degree. C. As
fibrin cross-linking relies on the catalytic action of thrombin,
fibrin-alginate components were separated into two streams and
combined above the T-junction (see, e.g., FIG. 23A). The acid was
added after the emulsion formed in the fabrication of
fibrin-alginate hydrogel capsules, as cross-linking of alginate
prior to fibrin cross-linking resulted in phase separation of the
two polymers. The hybrid emulsions were incubated at 37.degree. C.
to allow for the protein component to cross-link before the
emulsion was broken.
[0349] Confocal images were taken of cell-encapsulating hybrid
hydrogel capsules to assess the structure and distribution of
components within each hydrogel capsule. Microscopy revealed
collagen and fibrin fibrils within their respective hydrogel
capsules (FIG. 19A-B). The pixel intensities of the alginate
component of collagen-alginate hydrogel capsules followed a
unimodal distribution (FIG. 19C), indicating that there was no
phase separation of the two components. The collagen and alginate
components per hydrogel capsule were quantified (FIG. 19D). The two
components followed unimodal distributions, with a CV of 8.6% for
alginate and 24% for collagen.
[0350] The histogram of pixel intensities of the alginate component
of fibrin-alginate hydrogel capsules (FIG. 19E) exhibited greater
spread than that of collagen-alginate hydrogel capsules, with a
tail towards lower intensity values. This was reflected in the
confocal image of fibrin-alginate hydrogel capsules, in which the
alginate component was visibly heterogeneous (FIG. 19B). When
averaged per 10 pixels, corresponding to 4 microns, the
distribution of alginate intensities exhibited a tighter peak,
indicating that the variation in polymer concentration occurred on
a length scale of 4 microns (FIG. 19F). Quantification of the
fibrin and alginate components per hydrogel capsule revealed
greater variance than in collagen-alginate hydrogel capsules, with
11% of the hydrogel capsules lacking a fibrin component (FIG. 19G).
The cross-linking integrity of the protein component of both hybrid
hydrogel capsules was tested by exposure to
ethylenediaminetetraacetic acid (EDTA), a calcium chelator (FIG.
19G-H). Collagen-alginate hydrogel capsules exposed to EDTA
underwent a 10% decrease in intensity in the remaining collagen
portion and a 46% reduction in the number of distinct hydrogel
capsules, either through disintegration or combination with other
hydrogel capsules. Remaining hydrogel capsules retained their
fibrillar structure but lost their circular morphology (FIG. 23B).
Fibrin-alginate hydrogel capsules experienced only a 16% reduction
in number, and retained their circular morphology (FIG. 23C).
Example 11: Constructing Defined Mesoscale Tissues to Study MSC
Osteogenic Differentiation
[0351] To assess the behavior of multiple encapsulated cells when
assembled together, microwells of PDMS were used to template and
culture hydrogel capsules (FIG. 20A). PDMS microwell diameters were
specified to range from 50-221 .mu.m and arranged such that, per
750.times.750 .mu.m area, the surface area of microwells remained
equal, so as to equalize the macroscopic density of seeded cells.
Encapsulated D1 cells were seeded into microwells by gravitational
sedimentation. The number of cells that settled in each microwell
depended on the microwell diameter, and ranged, on average, from
2.6 to 37 cells (FIG. 20B). Compared to larger microwells,
significantly fewer hydrogel capsules per well area settled in the
smallest 200 .mu.m-deep microwells, possibly due to more
inefficient packing of hydrogel capsules as the well diameter
approached the hydrogel capsule size. Cell retention in smaller
microwells was not significantly different between well depths.
However, as PDMS is non-adherent, microwells needed to be
sufficiently deep to prevent sample loss during washing and media
changes. 200 .mu.m-deep microwells retained significantly more
hydrogel capsules in the largest-diameter microwells than 100
.mu.m-deep microwells (FIG. 20C).
[0352] To assess osteogenesis and the relationship between
assemblage size and D1 osteogenic differentiation, encapsulated D1
cells were fixed and stained for alkaline phosphatase (ALP) after 6
days of culture in differentiation medium. Confocal imaging
revealed a combination of cells that had remained inside hydrogel
capsules, and cells that had egressed from the hydrogel capsules
and colonized the outside surfaces of the hydrogel capsules (FIG.
20D). ALP expression was observed in microwells of all sizes but
correlated with the number of hydrogel capsules per well (r=0.76)
(FIG. 20E).
Example 12: Intravenous Delivery of Hydrogel Encapsulated Cells
[0353] The small size of hydrogel capsules fabricated with this
technique permits, for the first time, the intravenous delivery of
hydrogel encapsulated cells. The large majority of clinical trials
currently administering MSCs to patients utilize i.v. infusion of
bare cells (Bailey et al., 2014., Nat. Biotechol. 32, 721-723).
While polymeric encapsulation by droplet extrusion was previously
explored for prolonging the delivery of multicellular clusters for
in vivo transplantation, a number of issues were reported (Orive et
al. 2003, Nat. Med. 9, 104-107; Ma et al., 2013, Adv. Healthc.
Mater. 2, 667-672; Pareta et al., 2014, Microencapsulation
Technology. In: Regenerative Medicine Applications in Organ
Transplantation. Edited by Orlando G., Lerut J., Soker S. &
Stratta, R. J. 1st Ed. Academic Press, Boston, pages 627-635)
including that the large microparticles >100 .mu.m generated
with previous methods (Karoubi, 2009, Biomaterials 30, 5445-5455)
precluded i.v. infusion. To first determine if the hydrogel
capsules could retain structural integrity in circulation, they
were subjected to shear forces 10 times higher than arterial
pressure (.about.2 Pa). Even soft (E=.about.300 Pa) hydrogel
capsules were found to withstand this level of shear (FIG. 24A). To
explore the utility of this approach in improving delivery of
allogeneic donor cells, mMSCs derived from Balb/c mice were
injected into C57/BL6 mice. Consistent with previous observations
(Fischer et al. 2009, Stem Cells Dev. 18, 683-691), mMSCs were
localized in lungs shortly after the i.v. injection, likely due to
their entrapment into small capillaries (.about.3 .mu.m) (FIG.
25A). Strikingly, the half-life of donor cell clearance was
increased by .about.10 fold when cells were encapsulated, but not
when cells were mixed with empty hydrogel capsules (FIG. 25A, 25B).
This finding indicates that encapsulation of allogeneic donor cells
dramatically improves their maintenance in vivo after i.v.
infusion. Cell-free hydrogel capsules demonstrated similar
maintenance, suggesting that the durability of the encapsulating
hydrogel may set the upper limit to the survival of the
encapsulated cells. While MSCs are considered immunoprivileged,
some studies have shown that allogeneic transplantation leads to
immune rejection of donor MSCs (Eliopoulos et al., 2005, Blood 106,
4057-4065; Nauta et al., 2006, Blood 108, 2114-2120); encapsulation
in these thin gels can enable cells to bypass rejection. Human MSCs
(hMSC) encapsulated and injected in the immunocompromised
NOD/SCID/IL2.gamma..sup.-/- (NSG) mice yielded similar results,
with hydrogel encapsulation even enhancing the bioluminescence
signal after a day of injection in this model (FIG. 24B-C).
[0354] Studies were next performed to test whether prolonged cell
delivery was accompanied by sustained systemic presence of soluble
factors from allogeneic donor cells. mMSCs were genetically
modified to express Gaussia luciferase (Gluc, .about.20 kD), which
is constitutively secreted (Wurdinger, 2008, Nat. Methods 5,
171-173). Intravenous injection of encapsulated cells led to a
progressive increase in blood Gluc levels, with a peak at week 2
(FIG. 25C). In contrast, injection of unencapsulated cells led to
much lower blood levels of Gluc; the total amount of Gluc in blood
over 2 weeks was increased by .about.10 fold with encapsulation. To
evaluate whether this approach also results in sustained systemic
secretion of native soluble factors from donor cells, singly
encapsulated hMSCs were i.v. injected into NSG mice. The
encapsulation procedure did not compromise IL-6 secretion by human
MSCs either constitutively or under stimulation by IFN.gamma. (FIG.
24D). Injection of cells encapsulated in the higher, 232-kDa MW
polymer increased the total concentration of human IL-6 in blood
plasma at 24 hours by .about.2 fold, as compared to unencapsulated
cells (FIG. 25D). Interestingly, encapsulation of cells in the
lower, 139-kDa MW polymer led to no differences in plasma levels of
human IL-6 (FIG. 24E); as cells in hydrogel capsules fabricated
from lower MW polymers showed greater egress in vitro following
encapsulation, this result demonstrates that hMSCs encapsulated in
this polymer may have egressed and experienced immune clearance by
the host.
Example 13: Matrix Stiffness as a Modulator of Soluble Factor
Secretion from Encapsulated Cells
[0355] Secretion of soluble factors from the cells encapsulated in
the hydrogel capsules of the invention may be modulated by changing
the stiffness of the hydrogel capsules. Human primary bone marrow
MSCs were encapsulated in alginate hydrogels containing RGD
cross-links for cell attachment and characterized by different
stiffness (soft=0.3.about.1 kPa; stiff=>30 kPa). The MSCs were
cultured in media containing DMEM, 1% GlutaMax and 1% Penn/Strep in
the presence or absence of exogenous inflammatory factors
(INF-.gamma., TNF-.alpha. and LPS). The media were collected at 24,
48 and 72 hours and subjected to ELISA assays in order to evaluate
the total quantity of secreted factors, and the results are shown
in FIGS. 26A, 26B and 26C. Specifically, the results shown in FIG.
26A demonstrate that MSCs encapsulated in soft hydrogels secrete a
greater amount of IL-6 than MSCs encapsulated in stiff hydrogels,
and that stimulation with INF-.gamma. further increases the amount
of the secreted IL-6 in a dose-dependent manner. The results shown
in FIG. 26B demonstrate that MSCs encapsulated in soft hydrogels
secrete a greater amount of MCP-1 (CCL2) chemokine than MSCs
encapsulated in stiff hydrogels and that stimulation with
TNF-.alpha. further increases the amount of the secreted MCP-1 over
time. The results shown in FIG. 26C, left panel, demonstrate that
MSCs encapsulated in stiff hydrogels secrete a greater amount of
SCF over time, and that stimulation with LPS further increases the
amount of the secreted SCF over time. Finally, the results shown in
FIG. 26C, right panel show that MSCs encapsulated in soft hydrogels
secrete a greater amount of TGF-.beta. than MSCs encapsulated in
stiff hydrogels, in the absence of further stimulation by exogenous
factors.
[0356] For luciferase experiments, mouse D1 MSCs were transduced
with a DNA plasmid containing the DNA encoding the secreted form of
luciferase (Gaussia Luciferase, .about.20 kDa) and CFP gene
sequences (as intracellular control). In the plasmid, both genes
were separated by IRES, and their expression was driven by the
constitutive promoter SV40, as shown in the schematic in FIG. 27A.
The transduced cells were encapsulated in soft (0.3.about.1 kPa),
medium (10.about.20 kPa), and stiff (>30 kPa) alginate hydrogels
that contain an RGD sequence for cell adhesion. The total amount of
the secreted Gaussia Luciferase was evaluated at 24, 48 and 72
hours by adding coelenterazine to each well that contains samples
and integrating flash kinetics for 10s. The results shown in FIG.
27B demonstrate that D1 cells encapsulated in stiff hydrogels
secrete greater amounts of luciferase over time than D1 cells
encapsulated in soft or medium hydrogels. The results shown in FIG.
27C demonstrate that the total luciferase secretion is partially
inhibited by the addition of myosin-II inhibitor blebbistatin to
the cells, and that the inhibitory effect of blebbistatin is more
pronounced in stiff hydrogels as compared to soft and medium
hydrogels.
EQUIVALENTS
[0357] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
Sequence CWU 1
1
1314PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Arg Gly Asp Ser124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Arg
Glu Asp Val134PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 3Arg Gly Asp Val145PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Leu
Arg Gly Asp Asn1 555PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Ile Lys Val Ala Val1 565PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Tyr
Ile Gly Ser Arg1 575PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 7Pro Asp Ser Gly Arg1 5810PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Arg
Asn Ile Ala Glu Ile Ile Lys Asp Ala1 5 1094PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Arg
Gly Asp Thr1104PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 10Asp Gly Glu Ala1114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(3)..(3)Any amino acid 11Val Thr Xaa
Gly1129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Gly Gly Gly Gly Arg Gly Asp Ser Pro1
51311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Gly Gly Gly Gly Arg Gly Ala Ser Ser Lys Tyr1 5
10
* * * * *