U.S. patent application number 14/513893 was filed with the patent office on 2015-02-12 for microencapsulation process of sertoli cells, microcapsules obtained and use for treatment of type i diabetes mellitus.
The applicant listed for this patent is GH CARE, INC. D/B/A ALTUCELL, INC., GH CARE, INC. D/B/A ALTUCELL, INC.. Invention is credited to Ennio Becchetti, Riccardo Calafiore, Mario Calvitti, Francesca Fallarino, Giovanni Luca, Claudio Nastruzzi, Paolo Puccetti.
Application Number | 20150044109 14/513893 |
Document ID | / |
Family ID | 40929607 |
Filed Date | 2015-02-12 |
United States Patent
Application |
20150044109 |
Kind Code |
A1 |
Calafiore; Riccardo ; et
al. |
February 12, 2015 |
MICROENCAPSULATION PROCESS OF SERTOLI CELLS, MICROCAPSULES OBTAINED
AND USE FOR TREATMENT OF TYPE I DIABETES MELLITUS
Abstract
An apparatus for production of microcapsules includes a
volumetric pump configured to deliver a polysaccaridic suspension
through a catching tube. A needle-type element is configured to
receive the suspension through the catching tube, and the
needle-type element has a button hole opening in a lateral wall
thereof and an output hole that outputs the suspension. The button
hole opening receives a pressure fluid current. A pressure
regulator is coupled to the button hole opening to regulate the
pressure fluid current to interrupt a suspension flow and obtain
microdroplets of homogeneous size exiting the output hole. The
microdroplets are received in a receiving container in a solution
including divalent cations or polycationic substances to form a gel
such that homogeneous microcapsules are formed.
Inventors: |
Calafiore; Riccardo; (Ponte
Rio PG, IT) ; Luca; Giovanni; (Paola CS, IT) ;
Calvitti; Mario; (Perugia, IT) ; Becchetti;
Ennio; (Perugia, IT) ; Puccetti; Paolo;
(Perugia, IT) ; Fallarino; Francesca; (Perugia,
IT) ; Nastruzzi; Claudio; (Pontegradella Fe,
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GH CARE, INC. D/B/A ALTUCELL, INC. |
Dix Hills |
NY |
US |
|
|
Family ID: |
40929607 |
Appl. No.: |
14/513893 |
Filed: |
October 14, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13140728 |
Jun 17, 2011 |
8865218 |
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PCT/IB09/55847 |
Dec 18, 2009 |
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14513893 |
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Current U.S.
Class: |
422/243 |
Current CPC
Class: |
A61J 3/07 20130101; A61P
3/10 20180101; A61K 9/5036 20130101; A61K 9/5089 20130101; A61K
35/48 20130101 |
Class at
Publication: |
422/243 |
International
Class: |
A61J 3/07 20060101
A61J003/07 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2008 |
IT |
RM2008A000686 |
Claims
1.-13. (canceled)
14. An apparatus for production of microcapsules, comprising: a
volumetric pump configured to deliver a polysaccaridic suspension
through a catching tube; a needle-type element configured to
receive the suspension through the catching tube, the needle-type
element having a button hole opening in a lateral wall thereof and
an output hole that outputs the suspension, the button hole opening
receiving a pressure fluid current; a pressure regulator coupled to
the button hole opening to regulate the pressure fluid current to
interrupt a suspension flow and obtain microdroplets of homogeneous
size exiting the output hole, the microdroplets being received in a
receiving container in a solution including divalent cations or
polycationic substances to form a gel such that homogeneous
microcapsules are formed.
15. The apparatus as recited in claim 14, wherein the
polysaccaridic suspension includes a homogeneous suspension of
Sertoli Cells (SC) with purity, in terms of cell composition,
higher than 90%, in a saline solution of ultrapure sodium
alginate.
16. The apparatus as recited in claim 15, wherein the needle-type
element extrudes the suspension through the output hole to obtain a
continuous flow of microdroplets showing homogeneous
dimensions.
17. The apparatus as recited in claim 16, wherein the homogeneous
dimensions include the microcapsules with substantially fixed
diameters and without tails structures.
18. The apparatus as recited in claim 16, wherein the homogeneous
dimensions include the microdroplets having a mean diameter of
between about 300 and 700 microns with a standard deviation below
40 microns.
19. The apparatus as recited in claim 14, wherein the microcapsules
include sodium alginate, at a concentration of 1-3% w/v, with an
endotoxin content not exceeding 20 EU/g and a protein content lower
than 0.4%.
20. The apparatus as recited in claim 14, further comprising a
fluid generator coupled to a supply side of the pressure regulator
and having a pressure reduction to obtain a pressure fall of at
least 0.3 bars in a flow/no flow transient of fluid current.
21. The apparatus as recited in claim 19, wherein the fluid
generator includes a linear relationship between pressure cycles
and amount of fluid dispensed.
22. The apparatus as recited in claim 14, wherein the microcapsules
each include at least 20.times.10.sup.6 Sertoli Cells (SC).
23. The apparatus as recited in claim 14, wherein the microcapsules
including the Sertoli Cells are suitable as a sole therapeutic
agent for the prevention and treatment of Type 1 diabetes mellitus
(T1DM).
24. An apparatus for production of microcapsules including Sertoli
Cells, comprising: a volumetric pump configured to deliver a
polysaccaridic suspension through a catching tube, wherein the
polysaccaridic suspension includes a homogeneous suspension of
Sertoli Cells with purity, in terms of cell composition, higher
than 90%, in a saline solution of ultrapure sodium alginate; a
needle-type element configured to receive the suspension through
the catching tube, the needle-type element having a button hole
opening in a lateral wall thereof and an output hole that outputs
the suspension, the button hole opening receiving a pressure fluid
current wherein the needle-type element extrudes the suspension
through the output hole to obtain a continuous flow of
microdroplets showing homogeneous dimensions; a pressure regulator
coupled to the button hole opening to regulate the pressure fluid
current to interrupt a suspension flow and obtain microdroplets of
homogeneous size exiting the output hole, the microdroplets being
received in a receiving container in a solution including divalent
cations or polycationic substances to form a gel such that
homogeneous microcapsules are formed; and a fluid generator coupled
to a supply side of the pressure regulator and configured to supply
the pressure fluid current.
25. The apparatus as recited in claim 23, wherein the homogeneous
dimensions include the microcapsules with substantially fixed
diameters and without tails structures.
26. The apparatus as recited in claim 24, wherein the homogeneous
dimensions include the microdroplets having a mean diameter of
between about 300 and 700 microns with a standard deviation below
40 microns.
27. The apparatus as recited in claim 24, wherein the microcapsules
include sodium alginate, at a concentration of 1-3% w/v, with an
endotoxin content not exceeding 20 EU/g and a protein content lower
than 0.4%.
28. The apparatus as recited in claim 24, wherein the fluid
generator has a pressure reduction applied to obtain a pressure
fall of at least 0.3 bars in a flow/no flow transient.
29. The apparatus as recited in claim 28, wherein the fluid
generator includes a linear relationship between pressure cycles
and amount of fluid dispensed.
30. The apparatus as recited in claim 24, wherein the microcapsules
each include at least 20.times.10.sup.6 Sertoli Cells (SC).
31. The apparatus as recited in claim 24, wherein the microcapsules
including the Sertoli Cells are suitable as a sole therapeutic
agent for the prevention and treatment of Type 1 diabetes mellitus
(T1DM).
32. An apparatus for production of microcapsules including Sertoli
Cells, comprising: a container including a homogeneous suspension
of Sertoli Cells with a higher than 90% purity, in terms of cell
composition, in a saline solution of ultrapure sodium alginate; a
volumetric pump configured to aspirate the suspension to create a
suspension flow; a needle-type element configured to receive the
suspension flow; a pressure regulator coupled to the needle-type
element and configured to regulate a pressure fluid current
therethrough to interrupt the suspension flow and obtain
microdroplets of homogeneous size exiting an output hole of the
needle-type element, the microdroplets being received in a
receiving container in a solution including divalent cations or
polycationic substances to form a gel such that homogeneous
microcapsules are formed; and a fluid generator coupled to a supply
side of the pressure regulator and configured to supply the
pressure fluid current.
33. The apparatus as recited in claim 32, wherein the microcapsules
have substantially fixed diameters without tails structures and
have a mean diameter of between about 300 and 700 microns with a
standard deviation below 40 microns.
Description
[0001] The invention relates to the use of Sertoli cells (SC)
microencapsulated into hydrogel-based microcapsules, for the
prevention and/or treatment of Type 1 diabetes mellitus (T1DM) and
to a process for producing microcapsules, preferably shaped as
microspheres. The product object of the invention is capable of
inducing both the neogenesis of beta-cells, destroyed by the
diabetic pathology, and the "cutting off" of the same autoimmune
process responsible for such destruction in T1DM.
[0002] The treatment with microencapsulated SC allows preventing
and treating T1DM without resorting to any transplantation of
hexogen pancreatic islets (either human or animal). It should be
noted that the product obtained from SC microencapsulation is fully
comparable to a "conventional" drug.
STATE OF THE ART
[0003] The worldwide current incidence of type 1 diabetes mellitus
(T1DM) is equal to about 30,000 new cases a year. At the basis of
type 1 DM pathogenesis which mainly but non exclusively affects
young people and teenagers, is the destruction of most
insulin-producing pancreatic beta-cells by an autoimmune mechanism.
In short, the organism loses the immune tolerance towards the
pancreatic beta-cells in charge of insulin production and induces
an immune response, mainly cell-mediated, associated to the
production of autoantibodies, which leads to the self-destruction
of beta-cells.
[0004] The current T1DM therapy, based on the administration of
hexogen insulin, tends to restore the glucide homeostasis as close
as possible to that observed in physiological conditions. Insulin
therapy, however, is not capable of reproducing the pulsating
rhythm of insulin secretion typical of normal beta-pancreatic cell
in response to secretagogue stimuli.
[0005] The restoration of a physiological and steady
endocrine-pancreatic function would therefore represent the final
goal for the radical solution of the pathology. To this end, new
strategies have been proposed, such as the transplantation of whole
pancreas or that of islets isolated from pancreas of human
donors.
[0006] The hexogen insulin therapy currently used does not
represent in any way the final therapy for treating T1DM. To
overcome this problem, approaches have long been proposed which
envisage the transplantation of the entire pancreatic organ or that
of islets separated from the pancreas of human or animal
donors.
[0007] The transplantation of islets, compared to that of the whole
pancreas, is less invasive but exhibits similar problems, and in
particular:
1. Reduced availability of human pancreas from dead donors, and, as
a consequence, of islets. 2. Need of subjecting the recipient to
lifelong general pharmacological immunosuppression regimes. Such
therapeutic option used to prevent the immune rejection of the
transplanted tissue, however, is burdened by side effects that are
still little known nowadays, but also potentially very serious. 3.
Rejection of transplants of heterologous islets, since none of the
immunosuppressive drugs currently used has proved capable of
effectively preventing them. 4. Poor survival of the transplanted
islet tissue over time.
[0008] The Sertoli cell (SC) has recently been revaluated in its
functions and promoted from a mere structural support of the
testicular seminiferous tubule to a real biochemical laboratory
with countless trophic and immunological functions. In particular,
it has been proved that SC cultures produce molecules that inhibit
the proliferation of B and T lymphocytes (1). Moreover, to
strengthen their immunoregulatory function, the SC can induce the
apoptosis of T, B cells and natural killers, linking through their
ligand FAS to the FAS expressed by the target cells (2).
[0009] Another mechanism through which the SC carry out their
immunomodulating role is represented by the production of
Transforming Growth Factor-.beta. (TGF-.beta.) (3). This molecule
affects the phenotype of differentiation of T CD4+ lymphocytes,
favouring type Th2 (protective immunity) over type Th1 (non
protective immunity). As a whole, the Sertoli activity may
therefore have a direct clinical importance in T1DM, since beta
cells are destroyed by an infiltrate mainly consisting of
lymphocytes Th1 (INF-gamma positive).
[0010] The immunoregulating effect of SC, moreover, is associated
to the production of several growth factors, differentiating and
anti-apoptosis such as transforming growth factor
(TGF-.quadrature.), Glial Derived Neuroprophic Factor (GDNF),
interleukin-1 (IL-1), stem cell factor (cKit-ligand), Fas/Fas
Ligand (Fas-L), activin A and finally BCL-w (4).
[0011] The closest prior art (bibliographic reference No. 5)
describes the introduction of SC into ultrapure alginate
microcapsules with the obtainment of microcapsules with a mean end
diameter of 520.+-.14 .mu.m.
[0012] At the time of such article, at an international level,
microcapsules were considered satisfactory with a diameter of about
500 .mu.m and a percentage of "tails" not higher than 5%. Both the
capsular diameter and the presence of tails are very important
parameters. The first one, to be reduced as much as possible, to
allow a more effective exchange of metabolites; the second one as
it has recently been found that even a percentage of tails <5%
could trigger important phlogosis due to the creation of "loci
minoris resistentiae" wherein cellular antigens may be exposed.
[0013] The inventors of the present invention have surprisingly
found a process that allows producing homogeneous microcapsules of
smaller dimensions without the presence of tail structures that can
encapsulate SC while keeping their vitality and functionality
unaffected.
[0014] In consideration of the above, the invention proposes for
the first time the possibility of preventing and/or treating T1DM
by transplanting SC microencapsulated into hydrogel-based
microspheres, without any presence of hexogen islet tissue.
[0015] The object of the present invention therefore is a process
for the manufacture of hydrogel-based microcapsules, containing
Sertoli cells (SC) according to claim 1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Nine figures are attached to the present description, which
show:
[0017] FIG. 1. Microphotographs of swine SC. A:
Immunocytochemistry, obtained by incubating the preparation with
anti-mullerian inhibiting factor (MIS) antibodies. B:
Immunocytochemistry, obtained by incubating the preparation with
anti-vimentin antibodies. C-D: To prove the poor presence of Leydig
cells and peritubular cells, the preparation was subject to
histochemical techniques to assess the presence of alkaline
phosphatase, colouring with Fast-Red (typical of peritubular cells)
(C), and of 3-.beta.-hydroxy-steroidodehydrogenase activity,
colouring with Nitro-blue tetrazolium (typical of Leydig) (D).
[0018] FIG. 2. Apparatus for the production of SC microencapsulated
into alginate-based hydrogels through "air-monojet" (Panel A).
Panel B shows the most important components of the devised
system.
[0019] FIG. 3. Microphotographs of alginate-based microparticles
obtained by the atomising system "air monojet", using BaCl.sub.2
(A-C) and CaCl.sub.2+polyornithine (B-D) as gelling agents.
[0020] FIG. 4. Microphotographs in clear field of polysaccharidic
microparticles cross-linked with Barium (A) and Calcium (B) ions
after recovery from the peritoneal cavity of NOD rats 4 months
after the implant. Panel C shows a microphotograph obtained by a
fluorescence microscope of SC microencapsulated in barium alginate
after a dual colouring with EB/FDA.
[0021] FIG. 5. Percentages of spontaneous onset of T1DM (85%)
declared by the supplier of NOD rats (Taconic) (A), comparable to
those shown by the pre-diabetic animals of the "naive" control
group treated with empty microcapsules (B). On the other hand,
panel C shows how the pre-diabetic animals treated with
encapsulated SC have a percentage of onset of T1DM considerably
reduced, equal to just 9% (preventive effect).
[0022] FIG. 6. Values of post-transplant mean glycaemia in NOD rats
(Group E), with evident spontaneous diabetes treated with
microencapsulated SC (therapeutic effect).
[0023] FIG. 7. Analysis RT-PCR on splenocytes of animals treated
with microencapsulated SC. The results show that in animals from
Groups C and E (see section VII), the treatment with SC can
increase the number of positive in vivo Foxp3 cells. This result
denotes an important increase of T cells with regulating features,
that is, capable of regulating the activation and the proliferation
of several cells involved in immune responses.
[0024] FIG. 8. Histological analysis of pancreatic islets of
pre/diabetic NOD rats (A) and rats with spontaneous diabetes (B).
The images show that the islet is totally free from both perk and
intrainsular insulitic infiltrate. Panels C and D show the
histological analyses of islets of pre-diabetic "naive" NOD rats
(C) and suffering from spontaneous diabetes (D) treated with empty
capsules.
[0025] FIG. 9. Layout of the device according to the invention.
DESCRIPTION OF THE INVENTION
[0026] In the first place, a homogeneous polysaccharide suspension
of SC is produced: the solution has a 90% purity in terms of
cellular composition and is obtained in a saline solution of
ultrapure sodium alginate at a concentration comprised between 1
and 5% w/v, advantageously between 1 and 3%. The alginate used is
ultrapure as it exhibits an endotoxin content not higher than 20
EU/g and a protein content <0.4%; air is advantageously used as
fluid. SC are treated in advance with trypsin and EDTA (2 min), in
order to obtain a homogeneous cellular suspension. The following
were used to assess it: [0027] immunocytochemistry techniques,
incubating the preparation with anti-mullerian inhibiting factor
(MIS) and fluorescin anti-vimentin antibodies, which respectively
mark the MIS and vimentin molecules, both expressed by the SC only.
[0028] histochemical techniques to assess the presence of both
alkaline phosphatase (colouring with Fast-Red) typical of
peritubular cell, and of the 3-.beta.-hydroxy-steroidodehydrogenase
enzyme (colouring with Nitro-blue tetrazolium) which on the other
hand is typical of Leydig cells.
[0029] The results obtained with such histochemical assays have
allowed to prove the presence of 5-8% of peritubular and Leydig
cells; these cellular populations, moreover, are useful (when
present in these proportions) to ensure a molecular "cross-talk"
favourable for the correct functionality of SC.
[0030] This suspension is aspired at a speed comprised between 10
and 60 ml/min producing a continuous flow of dimensionally
homogeneous microdroplets through suction and extrusion using a
fluid current, advantageously air, at controlled pressure. The
suspension thus aspired is introduced in a needle-type element to
be divided into highly homogeneous microdroplets. Advantageously,
the needle-type element exhibits a buttonhole opening on the side
surface thereof wherein a fluid current is introduced at a rate of
3-7 litres/min to obtain a continuous flow of homogeneous size
microdroplets. The fluid current is obtained from a generator and
before being used, it is subject to a pressure reduction to obtain
a pressure drop in the flow-non flow transient not less than 0.3
Bar; to a regulation to obtain high reproducibility in the flow-non
flow transient and linearity between number of revolutions and
fluid current dispensed, and to a regulation and control of the
output current between 0 and 10 NL/min.
[0031] The microdroplets may have a mean diameter comprised between
300 and 700 .mu.m with a standard deviation below 40 .mu.m. The
microdroplets obtained are introduced in an aqueous solution,
advantageously using sterilised water for injectable preparations,
F.U, containing divalent cations or polycationic substances with
resulting gelification and obtainment of said microcapsules.
[0032] A further object of the present invention are Sertoli cells
as sole therapeutic agent for the prevention and radical cure of
T1DM.
[0033] Advantageously, according to the process of the invention,
the aspiration takes place continuously by a peristaltic pump at a
flow speed comprised between 10 and 16 ml/min and said extrusion
takes place through the "air monojet" system using a fluid flow,
preferably air, comprised between 3 and 7 l/min. In the process,
the exact calibration of said air flow, a characterising element of
the entire method, is ensured by the below components of the system
that are not present in previous methods (including that used in
the "Closest Prior Art"). Before coming into contact with said
suspension of said stage b) said air flow is subject to the
following operations with the following devices: [0034] the
membrane pressure reducer Swagelok (KPR1JRF411A20000) which is
capable of ensuring high reproducibility of the output pressure and
so as to obtain a very low pressure drop in the flow/non flow
transient below 0.3 Bar, serves for stabilising and making the air
flow to be sent to the extruder reproducible; [0035] regulation
through a micrometric valve Swagelok (SS-SS6MM), in output to the
pressure regulator, which allows regulating very finely the output
air flow (0-10 NL/min) with a high reproducibility in the flow/non
flow transient and maintaining linearity between number of
revolutions and dispensed flow; [0036] with rotating float flow
meter (ROTAMETRO) Yokogawa, supplied by Precision Fluid
(RAGK41-TOSS-SSNNN-M741A-TTCGN*A), located downstream of the
micrometric valve, which allows a precise and quick reading of the
output flow (0-10 NL/min) and thus the adjustment thereof through
the micrometric valve.
[0037] A further object of the present invention are microcapsules
containing SC obtainable according to the process of the invention,
one of the features thereof is to exhibit the secretion of IGF-1 of
microencapsulates SC identical to that of non-microencapsulates or
"free" SC.
[0038] A further object of the present invention is the use of
Sertoli cells, advantageously microencapsulated according to the
process of the invention, as sole therapeutic agent for the
production of a medicament of the prevention and radical cure of
T1DM.
[0039] According to the invention, the microcapsules obtained can
be subject to washing operations and/or further coating with
natural and/or artificial polymers.
[0040] Compared to the prior art, the process of the invention
allow a) producing microcapsules of smaller size, with fixed
diameters (starting from 300 .mu.m) and perfectly homogeneous,
without the presence of "tails" structures and above all, without
loss of vitality and functionality of the microencapsulated SC; b)
increasing the number of microencapsulated SC by ml of alginate
from 10.sup.6 SCs to 20.sup.6 SC by ml of alginate with imaginable
implications on the possibility of implanting a larger number of SC
in the smallest possible volume of polymer and c) increasing the
functionality of microencapsulated SC, in particular relating to
the production of IGF-1, the secretion thereof changes from 50
ng/ml/20.times.10.sup.6 cells) to 80 ng/ml/20.times.10.sup.6 cells
substantially equal to that of "free" SC.
With reference to the present invention, it should be noted that
[0041] 1. For the first time, microencapsulated SC are proposed as
final therapeutic approach, inducing the neogenesis of patient's
beta-cell, destroyed by the autoimmune process. [0042] 2. An
optimisation of the microencapsulation process has been obtained
which leads to the production of microcapsules with improved
features, such as the reduction of mean dimensions, the reduced
polydispersity and the absence of morphological deformities of the
microcapsules ("tails" and coalescences).
[0043] A further object of the present invention are compositions
comprising SC contained in microcapsules obtainable by the process
of the invention together with physiologically tolerable carriers
to use for the prevention and treatment of T1DM. An example of
carrier consists of saline for intraperitoneal administration.
Below are the detailed aspects of the present invention.
Purification of Polymers
[0044] The polymers usable for microencapsulating the SC are not
available on the market in the highly purified form strictly
necessary for applications requiring parenteral administrations,
such as human transplants. In these cases, in fact, strict
internationally recognised criteria of "quality control" are
required (see guidelines of the Ministry of Health and of U.S.
Pharmacopeia).
[0045] Most commercial products, in fact, have quite high endotoxin
levels (generally comprised between 30,000 and 60,000 EU/g) which
make them totally unsuitable for transplant procedures, which
require endotoxin levels not higher than 100 EU/g. As a consequence
of the above, all the polymers used for producing microparticles
are suitably subject to subsequent purification cycles that allow
the drastic reduction of the endotoxins present.
Isolation of SC
[0046] The SC may be isolated and purified from various animal
sources, generally prepuberals. After anaesthesia, the animals are
subject to bilateral orchiectomy. After the removal of the
epididymis, the testicles are subject to multienzymatic digestion.
Once the digestion is complete, the tubular tissue is subject to
filtration. The tubules thus obtained are placed in a culture at
37.degree. C. in a 5% atmosphere of CO2. After 48 hours in
incubator, the SC start adhering to the culture plates, forming a
cellular monolayer. The SC obtained are analysed in terms of
purity, vitality and functionality. The cellular vitality test is
routinely conducted immediately after the isolation, on the second
day of culture and immediately before and after the
microencapsulation process.
Production of Microencapsulated Sertoli Cells
[0047] The SC may be immobilised into microcapsules consisting of
various hydrogels consisting of hydrophilic polymers used alone or
in mixtures. The initial phase of the microencapsulation process
envisages the obtainment of a continuous and calibrated flow of
microdroplets. Various procedures may be used for obtaining the
microdroplets: (a) "air-monojet" microencapsulator, (b) automatic
vibrating encapsulator, (c) electrostatic microencapsulator e (d)
microfluidic lab-on-a-chip systems.
[0048] Once a flow of microdroplets with controlled and homogeneous
dimensions is obtained, these are transformed into solid
microspheres through gelification procedures. For example:
converging monolayers of SC are treated to obtain a homogeneous
cellular suspension, the SC are resuspended in the various
ultrapure polymeric solutions (obtained as described in section
"Purification of polymers") and finally, the microcapsules obtained
in the gelling bath are washed and isolated. The microcapsules
produced may be used as such or be further coated with various
natural, semi-synthetic or synthetic polymeric layers. The method
proposed therefore allows (as shown by the pictures of FIG. 3)
immobilising the SC into microcapsules with highly homogeneous
dimensions, without morphological defects (presence of coalescences
or "tail" structures), ensuring that the vitality and functionality
features of the encapsulated cells are maintained.
In Vivo Biocompatibility of Encapsulated SC
[0049] The microparticle biocompatibility is assessed through the
intraperitoneal implant carried out through abdominal incision. The
body weight of each recipient animal is monitored during all the in
vivo study. At different times from the transplant, the
microcapsules are explanted to assess their morphology and function
of the encapsulated cells. The general features of the recovered
microspheres were determined through microscopic analysis,
assessing both the morphology and any presence of inflammatory
cells of the capsule surface. The vitality of microencapsulated SC
was also assessed using the dual colouring technique with
EB/FDA.
Assessment of the In Vivo Activity of Microencapsulated SC
[0050] It has been proved that the intraperitoneal transplant of
microencapsulated SC in saline is capable of both preventing and
treating T1DM in "stringent" animal models of human T1DM, such as
NOD rats. Advantageously, but not exclusively, the administration
of the product obtained from the microencapsulation of SC according
to the invention takes place by intraperitoneal administration,
with the product carried in saline.
[0051] A further object of the invention is a device for producing
microcapsules advantageously for applying the process of the
invention. The device and the operation thereof shall now be
described with reference to FIG. 9. A first container 2 cooperates
with flow dispensing means 4, advantageously a volumetric pump, for
delivering suspension 1 through the catching tube 3 to a
needle-type element 5. The needle-type element 5 exhibits a
buttonhole opening 6 in the lateral wall thereof and output hole 7.
A joint 8 allows a pressure fluid current 10, preferably air,
coming from a generator 9 and regulated by adjusting means 11, to
enter inside element 5. By suitably regulating current 10 it is
possible to interrupt the suspension flow and obtain microdroplets
13 of homogeneous size, which form gel in a solution containing
divalent cations present in a second container 12. The airjet
instrument mentioned above and the conditions described are applied
for obtaining the homogeneous microcapsules.
Development of a Prototype of Microencapsulator Usable in Sterility
Conditions and GLP
EXAMPLES
Microencapsulation of Sertoli Cells Into Alginate-Based
Microspheres and Assessment of the In Vivo Biocompatibility and
Functionality
Purification of the Polymer
[0052] Sodium alginate obtained through a process of sequential
filtrations, was used as base polymer for the production of
microcapsules, usually available in a 1-6% (w/v) solution,
appropriately stored in a place protected from light and at a
temperature of 4.degree.-6.degree. C. Said compound is stable over
time for about 5 years, has an endotoxin content not higher than 20
EU/g and a virtually absent protein content (<0.4%--another
criterion of "bioinvisibility" of U.S. FDA).
Isolation of SC from Prepuberal Baby Swine
[0053] The SC were isolated from testicles of baby swine (7-15 days
old) "Large-White". After anaesthesia through the i.m.
administration of 0.1 mg/kg azaperon (Stresnil.RTM. 40 mg/ml,
Janssen, Brusselle, Belgium) and 15 mg/kg ketamine (Imalgene.RTM.
100 mg/ml, Gellini Farmaceutici, the swine were subject to
bilateral orchiectomy. After the removal of the epididymis, the
testicles are decorticated from the albuginea, finely chopped into
small tissue fragments (1-3 mm3) and immediately subject to a first
enzymatic digestion based on collagenase P (Roche Diagnostics,
S.p.A., Monza, Italy) (2 mg/ml) in HBSS (Sigma Chemical Co, St.
Louis, USA). The digestion is continued up to the separation of the
seminiferous tubules. The collected tubules are then washed in HBSS
and centrifuged at 500 r.p.m. After the wash, the tubules are
incubated with a solution of HBSS containing trypsin (2 mg/ml) and
DNAse I (Sigma). After the completion of the second digestion, the
trypsin solution is diluted 1:1 with Hank's+20% FBS to stop the
enzymatic activity thereof. After further washes with HBSS, the
tubules are separated from the peritubular cells through a light
centrifugation at 300 rpm. The "pellet" containing the tubular
tissue is suitably filtered with a stainless steel filter with a
500 .mu.m mesh opening. Finally, in order to remove any peritubular
and Leydig cells contaminating the preparation, the suspension
containing the tubules is further centrifuged at 800 rpm for 5 min
and the resulting pellet is treated for 7 min with a glycine 1 M
solution and EDTA 2 mM in HBSS at pH 7.2.
[0054] The tubules thus obtained are placed in a culture in HAM F12
(Euroclone) supplemented with retinoic acid 0.166 nM (Sigma) and
with 5 ml/500 ml insulin-transferrin-selenium (ITS) (Becton
Dickinson#354352), at 37.degree. C. in a 5% atmosphere of CO2.
After 48 hours of culture, the SC start adhering to the culture
plates, forming a cellular monolayer. In order to remove the
residual germ cells (which, as known, if implanted in the
peritoneal cavity may give rise to dysgerminoms), the SC monolayers
are treated with a buffer, tris-(hydroxymethyl)-aminomethane
hydrochloride (TRIS) (Sigma) that allows eliminating the residual
germ cells through osmotic lysis. Finally, the SC are grown in the
above conditions, usually in 75 cm2 flasks.
[0055] The SC obtained were analysed in terms of purity, vitality
and functionality. The purity of the SC, which was higher than 90%,
was assessed by immunocytochemistry techniques, incubating the
preparation with anti-mullerian inhibiting factor (MIS) and
fluorescin anti-vimentin antibodies, which respectively mark the
MIS and vimentin molecules, both expressed by the SC only (FIG. 1
A, B).
[0056] To prove the reduced presence of Leydig and peritubular
cells as possible contaminants, the SC preparations were subject to
histochemical assessments. These tests allow assessing both the
presence of alkaline phosphatase (colouring with Fast-Red) typical
of peritubular cell, and of the
3-.beta.-hydroxy-steroidodehydrogenase enzyme (colouring with
Nitro-blue tetrazolium) which on the other hand is typical of
Leydig cells (FIG. 1 C, D). The results obtained with these
histochemical assays have allowed to prove the presence of 5-8% of
peritubular and Leydig cells; these cellular populations, moreover,
are useful (when present in these proportions) to ensure a
molecular "cross-talk" favourable for the correct functionality of
these populations of testicular cells.
[0057] The vitality of SC was determined by treatment with ethidium
bromide (EB) and fluorescein-diacetate (FDA) (Sigma). The cells,
observed by a fluorescence microscope, in all conditions showed a
vitality higher than 95%. The cellular vitality test is routinely
conducted immediately after the isolation, on the second day of
culture and immediately before the microencapsulation process.
C) Production of Microdroplets for Encapsulating Sertoli Cells
[0058] The SC were immobilised into microcapsules consisting of
various polysaccharide polymers used alone or in mixtures. The
selected polymer was sodium alginate ultrapurified at our
laboratories. The initial phase of the microencapsulation process
envisages the obtainment of a continuous and calibrated flow of
microdroplets starting from cellular suspension of SC in an aqueous
polysaccharide suspension with a polymeric concentration variable
between 1 and 5% (w/v).
[0059] Various procedures were and may be used for obtaining the
microdroplets: (a) "air-monojet" microencapsulator, (b) automatic
vibrating encapsulator, (c) electrostatic microencapsulator e (d)
microfluidic lab-on-a-chip systems.
[0060] In particular, the method selected (a), based on a
semi-automatic, compact, sterilisable and transportable
microencapsulator (FIG. 2, A shows an overall view of the system),
has allowed producing microcapsules containing SC with highly
homogeneous dimensions (300 to 700 .mu.m diameter), without any
evident morphological flaw (such as the presence of coalescences or
"tail" structures) and above all, without the loss of vitality and
functionality of the microencapsulated SC.
[0061] Panel B of FIG. 2 schematises the procedure of the
microencapsulation process through "air-monojet".
D) Preparation of Ultrapurified Alginate-Based Microcapsules
[0062] Once a flow of microdroplets with controlled and homogeneous
dimensions is obtained, these are transformed into solid
microspheres through a gelification procedure which envisages the
forming of ionic links with divalent ions according to a method
developed and validated at our laboratories.
[0063] In particular, converging monolayers of SC are treated with
0.05% trypsin/EDTA (Gibco, Grandisland, USA) (2 min), in order to
obtain a homogeneous cellular suspension. Once washed, the SC are
counted by hemocytometric analysis and tested for vitality.
Afterwards, the SC are resuspended in the various ultrapure
polymeric suspensions in concentrations variable between 1.5-2%
(w/v) of AG. For the production of microcapsules with the
"air-monojet" system, the SC suspension in the polymers is
continuously aspired by a peristaltic pump at a flow speed
comprised between 10 and 16 ml/min. The cellular suspension is then
extruded through the "air monojet" system (using an air flow
comprised between 3 and 7 l/min). During the entire process, the SC
suspension is kept under light stirring to prevent the cellular
aggregation and the possible formation of microcapsules with
non-homogeneous distribution of SC therein.
[0064] The microdroplets produced are gellied with a solution
containing divalent cations, such as Ca+2 or Ba+2 (0.5-2.5%, w/v).
In this way, the microdroplets are instantly transformed into gel
microspheres. Afterwards, the microcapsules are left to settle for
periods variable between 2 and 15 min into the gelling bath. At the
end of this step, the microcapsules are subject to repeated washing
cycles with saline.
[0065] The microcapsules produced may be used as such or be further
coated through sequential incubation in solutions containing
natural, semi-synthetic or synthetic cationic polymers. For
example, poly-L-ornithine (PLO) was used at 0.12% (for 10 min) and
at 0.06% (for 6 min). Finally, the microcapsules coated with PLO
are further treated with a diluted solution of polysaccharide, to
provide the highly biocompatible final outer coating.
[0066] FIG. 3 shows the microphotographs of alginate-based
microcapsules obtained by the procedure described above, both using
only Barium ions (A-C) and the procedure of the multiple coating
with Calcium/polyornithine/polymer ions (B-D). The method proposed
therefore allows (as shown by the pictures of FIG. 3) immobilising
the SC into microcapsules with highly homogeneous dimensions,
without morphological defects, such as the presence of coalescences
or "tail" structures, and finally, ensuring that the vitality and
functionality features of the encapsulated cells are
maintained.
E) In Vivo Biocompatibility of Encapsulated SC
[0067] After general anaesthesia, induced by intra-peritoneal
administration of 100 mg/kg ketamine (Parke-Davis/Pfizer,
Karlsruhe, Germany) and 15 mg/kg xylazine (Bayer, Leverkusen,
Germany), the alginate microparticles were introduced through a
small abdominal incision in the peritoneal cavity of female NOD
rats (Harlan, Italy, approximate weight of 25 g). 106
microencapsulated SC were implanted in each animal. The body weight
of each recipient rat was monitored during all the in vivo
study.
[0068] After 4 months from the transplant, the microcapsules were
explanted, after anaesthesia, from the peritoneal cavity of the
animals to assess their morphology and function of the contents.
The microcapsules were recovered by peritoneal wash using saline.
The general features of the recovered microspheres were determined
through microscopic analysis, assessing both the morphology and any
presence of inflammatory cells of the capsule surface. The vitality
of microencapsulated SC was also assessed using the dual colouring
technique with EB/FDA.
[0069] The microphotographs shown in FIG. 4 (A-B) show that the
polysaccharidic microparticles keep high biocompatibility
standards, as shown by the minimum levels of inflammatory cells
present on the capsular surface. Moreover, the microencapsulated SC
both in Barium (FIG. 4A) and calcium (FIG. 4B) alginate, keep
excellent vitality levels 4 months after the implantation (FIG. 4
C).
E) Assessment of the In Vivo and In Vitro Activity of
Microencapsulated SC
[0070] The present invention finds application in the field of
transplantation biotechnologies, such as for example the prevention
and treatment of T1DM. Actually, at our laboratories we have proved
that the intraperitoneal transplant of microencapsulated SC in
barium alginate microspheres (206/rat) is capable of both
preventing and treating T1DM in "stringent" animal models of human
T1DM, such as NOD rats. In particular, SC microencapsulated into
barium alginate (BaAG) microspheres were transplanted, after 72
hours culture, in the peritoneal cavity of pre-diabetic NOD rats
and affected by evident diabetes. The implantation was carried out
in a general anaesthesia through laparotomy. The transplanted
animals were then monitored with weekly checks for their body
weight and glycaemia before and after meals. The experimental
protocol we followed envisaged groups of animals subject to
different treatments as indicated below.
[0071] Group A: "naive" pre-diabetic control animals (treated with
empty microcapsules).
[0072] Group B: control animals (treated with empty microcapsules)
with spontaneous diabetes.
[0073] Group C: "naive" pre-diabetic animals treated with
intraperitoneal implant of microencapsulated SC.
[0074] Group D: "naive" pre-diabetic animals treated with
intraperitoneal implant of "free" SC: (206/rat).
[0075] Group E: animals with spontaneous diabetes treated with
intraperitoneal implant of microencapsulated SC.
[0076] During the course of the in vivo study, some animals were
sacrificed to assess the peripheral immunological layout through
collection of spleen, peripancreatic lymph nodes and pancreas with
concurrent histomorphological and immunocytochemical
examinations.
[0077] The complete analysis of in vivo experiments on NOD rats has
allowed proving that microencapsulated SC transplanted in
pre-diabetic animals suffering from spontaneous diabetes allowed
obtaining, important therapeutic results, as shown below.
[0078] (A) Microencapsulated SC are capable of preventing the onset
of T1DM in NOD rats. This sensational result can be obtained from
the analysis of the percentages of spontaneous onset of T1DM. In
fact, this pathology occurred spontaneously in 85% of the animals
in group A (FIG. 5 B). This result is perfectly in line with the
percentages of occurrence of T1DM declared by the supplier of NOD
rats (Taconic) (FIG. 5 A).
[0079] On the other hand, in the animals of group C (the
pre-diabetic ones treated with encapsulated SC), the percentage of
onset of T1DM was only 9% (FIG. 5 C). Finally, in the animals of
group D (pre-diabetic treated with intraperitoneal implant of
"free" SC), the percentage of onset of T1DM was greatly reduced
compared to that of the "naive" (19%), although higher than in the
animals of group C (FIG. 5 D).
[0080] (B) The microencapsulated SC are capable of normalising, in
just 7-15 days from the implant, the glycaemic values (with the
attainment of glycaemia below 200 mg/dl) in more than 60% of rats
in Group E (N=30) that had spontaneously developed diabetes (FIG. 6
A). On the other hand, the animals in Group B (diabetics treated
with empty capsules) always kept high levels of glycaemia, dying
quickly, in 1-2 weeks. Finally, the animals in group F (N=30)
(diabetics treated with intraperitoneal implant of "free" SC) were
able to normalise the glycaemic values although in a lower
percentage, equal to about 40%.
[0081] (C) Studies carried out on lymph nodes, pancreas and spleens
have shown that the SC are capable of "re-educating" the immune
system of the animals in Groups C and E, "blocking" the autoimmune
attack responsible for the disease, as can be seen from FIG. 7
relating to real time Polymerase chain reaction (PCR) results on
the splenocytes of treated animals.
[0082] In particular, such results show that one of the main
effects of the treatment with SC is their capacity to induce in
vivo Foxp3+ cells. Foxp3 is a selective marker of T cells with
regulating features, that is, capable of regulating the activation
and the proliferation of several cells involved in immune responses
and the number whereof is reduced in the NOD rat model.
[0083] (D) Histochemical assays carried out on all the groups of
animals studied show that the treatment with SC is capable of
removing the insulitic mononuclear infiltrate at the pancreas level
in animals of groups C and E compared to those of the control
groups (A and B) (FIG. 8). Moreover, such effect was followed by
the activation of pancreatic mesenchymal stem cells capable of
generating new .beta.-cells which are capable of normalising
glycaemia in animals treated with SC, as they are not undermined by
the autoimmune attack anymore. The remodulation of the immune
response after treatment with SC is mediated by the activation of
the immunoregulatory pathway of the indoleamine 2 3-dioxygenase
(IDO) enzyme, an isoform whereof is expressed and functioning in
SC, too.
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