Cryopreserved Implantable Cell Culture Devices and Uses Thereof
The invention provides cryopreserved encapsulated cell therapy devices that are capable of delivering biologically active molecules as well as methods of using these devices.
1 . A method of cryopreserving an implantable cell culture device, the device comprising:
a) a core comprising
(i) a cell line comprising an ARPE-19 cell genetically engineered to produce a therapeutically effective amount of one or more cytokines, neurotrophic factors, soluble receptors, anti-angiogenic antibodies and molecules, or biologically active molecules that are introduced into the ARPE-19 cell by an iterative transfection process, wherein the iterative transfection comprises one transfection, two transfections, or three transfections,
(ii) a cell line comprising an ARPE-19 cell genetically engineered to produce a therapeutically effective amount of one or more cytokines, neurotrophic factors, soluble receptors, anti-angiogenic antibodies and molecules, or biologically active molecules that at least 10,000 ng/day/10 6 cells; or
(iii) ARPE-19 cells genetically engineered to secrete a therapeutically effective amount one or more biologically active molecules, and
b) a semi-permeable membrane surrounding the cell line in (i), the cell line in (ii), or the ARPE-19 cells in (iii), wherein the membrane permits the diffusion of the cytokines, neurotrophic factors, soluble receptors, anti-angiogenic antibodies and molecules, or biologically active molecules there through,
the method comprising the steps of:
adding a cryoprotectant agent to the core of the device,
placing the device in a cryogenic storage vial,
freezing the devices under controlled rate freezing, and
storing the device in dry ice (−70° C.), in a freezer (−80° C.), in vapor phase liquid nitrogen (−190° C.), or any combination thereof.
2 . The method of claim 1 , wherein the cryoprotectant agent is 10% glycerol.
3 . The method of claim 21 , wherein the controlled rate freezing occurs at −80° C.
4 . The method of claim 31 , wherein the method further comprises the step of transporting the devices under vapor phase liquid nitrogen (−190° C.) conditions, under dry ice (−70° C.) conditions, or a combination thereof.
5 . The method of claim 1 , wherein the cell line in (i) produces between 10,000 and 30,000 ng/day/10 6 cells of the one or more cytokines, neurotrophic factors, soluble receptors, anti-angiogenic antibodies and molecules, or biologically active molecules, when the iterative transfection is one transfection.
6 . The method of claim 5 , wherein the cell line in (i) produces about 15,000 ng/day/10 6 cells of the one or more cytokines, neurotrophic factors, soluble receptors, anti-angiogenic antibodies and molecules, or biologically active molecules.
7 . The method of claim 1 , wherein the cell line in (i) produces between 30,000 and 50,000 ng/day/10 6 cells of the one or more cytokines, neurotrophic factors, soluble receptors, anti-angiogenic antibodies and molecules, or biologically active molecules, when the iterative transfection is two transfections.
8 . The method of claim 7 , wherein the cell line in (i) produces about 35,000 ng/day/10 6 cells of the one or more cytokines, neurotrophic factors, soluble receptors, anti-angiogenic antibodies and molecules, or biologically active molecules.
9 . The method of claim 1 , wherein the cell line in (i) produces between 50,000 and 75,000 ng/day/10 6 cells of the one or more cytokines, neurotrophic factors, soluble receptors, anti-angiogenic antibodies and molecules, or biologically active molecules, when the iterative transfection is three transfections.
10 . The method of claim 9 , wherein the cell line in (i) produces about 70,000 ng/day/10 6 cells of the one or more cytokines, neurotrophic factors, soluble receptors, anti-angiogenic antibodies and molecules, or biologically active molecules.
11 . The method of claim 1 , wherein the one or more biologically active molecules is selected from the group consisting of neurotrophins, interleukins, cytokines, growth factors, anti-apoptotic factors, angiogenic factors, anti-angiogenic factors, antibodies and antibody fragments, antigens, neurotransmitters, hormones, enzymes, lymphokines, anti-inflammatory factors, therapeutic proteins, gene transfer vectors, brain derived neurotrophic factor (BDNF), NT-4, ciliary neurotrophic factor (CNTF), Axokine, basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF I), insulin-like growth factor II (IGF II), acid fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a (TGF α), transforming growth factor β (TGF β), nerve growth factor (NGF), platelet derived growth factor (PDGF), glia-derived neurotrophic factor (GDNF), Midkine, phorbol 12-myristate 13-acetate, tryophotin, activin, thyrotropin releasing hormone, interleukins, bone morphogenic protein, macrophage inflammatory proteins, heparin sulfate, amphiregulin, retinoic acid, tumor necrosis factor α, fibroblast growth factor receptor, epidermal growth factor receptor (EGFR). PEDF, LEDGF, NTN, Neublastin, VEGF inhibitors, other agents expected to have therapeutically useful effects on potential target tissues, and any combination(s) thereof.
12 - 13 . (canceled)
14 . The method of claim 1 wherein the core further comprises a matrix disposed within the semi-permeable membrane.
15 . The method of claim 14 , wherein the matrix comprises a plurality of monofilaments, wherein said monofilaments are
a. twisted into a yarn or woven into a mesh, or
b. twisted into a yarn that is in non-woven stands, and wherein the cells are distributed thereon.
16 . The method of claim 15 , wherein the monofilaments comprise a biocompatible material selected from the group consisting of acrylic, polyester, polyethylene, polypropylene polyacetonitrile, polyethylene terephthalate, nylon, polyamides, polyurethanes, polybutester, silk, cotton, chitin, carbon, and biocompatible metals.
17 . (canceled)
18 . The method of claim 1 , wherein the device further comprises a tether anchor.
19 . The method of claim 18 , wherein the tether anchor comprises an anchor loop.
20 . The method of claim 19 , wherein the anchor loop is adapted for anchoring the device to an ocular structure.
21 - 22 . (canceled)
23 . The method of claim 1 , wherein the semi-permeable membrane comprises a permselective, immunoprotective membrane.
24 . The method of claim 1 , wherein the semi-permeable membrane comprises an ultrafiltration membrane, a microfiltration membrane, or a non porous membrane material.
25 - 26 . (canceled)
27 . The method of claim 24 , wherein the non-porous membrane material is a hydrogel or a polyurethane.
28 - 29 . (canceled)
30 . The method of claim 1 , wherein the device is configured as a hollow fiber or a flat sheet.
31 - 33 . (canceled)
34 . The method of claim 1 , wherein at least one additional biologically active molecule is co-delivered from the device.
35 . The method of claim 34 , wherein the at least one additional biologically active molecule is from a non-cellular source or from a cellular source.
36 . (canceled)
37 . The method of claim 35 , wherein the at least one additional biologically active molecule is produced by one or more genetically engineered ARPE-19 cells in the core.
38 . The method of claim 1 , wherein the device further comprises one or more additional characteristics selected from the group consisting of:
a. the core comprises between 0.5-1.0×10 6 ARPE-19 cells;
b. length of the device is between 1 mm-20 mm;
c. the internal diameter of the device is between 0.1 mm-2.0 mm;
d. the ends of the device are sealed using methyl methacrylate;
e. the semi-permeable membrane has a median pore size of about 100 nm;
f. the nominal molecular weight cut off (MWCO) of the semi-permeable membrane is between 50 and 500 KD;
g. the semi-permeable membrane is between 90-120 μm thick;
h. the core comprises an internal scaffold, wherein the scaffold comprises polyethylene terephthalate (PET) fibers that comprise between 40-85% of the internal volume of the device; and
i. combinations thereof.
39 . The method of claim 38 , wherein the device comprises 2, 3, 4, 5, 6, 7, or all of the additional characteristics.
40 - 60 . (canceled)
61 . The method of claim 1 , wherein the cryopreserved device is thawed prior to implantation.
62 . The method of claim 61 , wherein following thawing, the device is implanted into the eye of a patient.
63 . The method of claim 62 , wherein the device is implanted in the vitreous, the aqueous humor, the Subtenon's space, the periocular space, the posterior chamber, or the anterior chamber of the eye.