Conformal coating of cells for immunoisolation
Hydrodynamic methods for conformally coating non -uniform size cells and cell clusters for implantation, thus preventing immune rejection or inflammation or autoimmune destruction while preserving cell functionality. A method for conformally coating cells and c clusters with hydrogels that are biocompatible, mechanically and chemically stable and porous, with an appropriate pore cut-off size. The methods of the invention are advantageously reproducible and result in a relatively high yield of coated versus non-coated cell clusters, without compromising cell functionality. Conformal coating devices configured to perform the methods of the invention, methods of optimally utilizing said devices and purifying the coated islets, and coated biomaterials made by said methods.
1. A method of treating a disorder in a patient, comprising implanting into the patient a biomaterial conformally coated by a method comprising the steps of:
a) injecting a water phase comprising a biomaterial and a coating material within a coaxial oil phase in a coating device that allows for a transition from dripping to jetting and flow elongation of the water phase within the oil phase;
wherein polymerization of the coating material occurs downstream of breakup of the water phase jet into particles, resulting in the conformal coating of the biomaterial with the coating material;
b) purifying the conformally coated biomaterial and biomaterial-free coating material from the oil phase; and
c) separating the conformally coated biomaterial from the biomaterial-free coating material;
wherein the biomaterial comprises cells or cell clusters; wherein the conformal coating around the biomaterial ranges from 1-100 μm in thickness, and wherein the coating is not a layer by layer coating directly on the cells or cell clusters.
2. The method of claim 1 , wherein said disorder is diabetes.
3. The method of claim 1 , wherein said conformally coated biomaterial comprises islet cells or cell clusters.
4. The method of claim 1 , wherein the conformally coated biomaterial comprises polyethylene glycol (PEG).
5. The method of claim 1 , wherein:
a) the water phase comprises 75,000 islet cells/ml, and further comprises:
i) 10% polyethylene glycol (PEG), 2% polyoxyethylene-polyoxypropylene block copolymer, and 0.62% w/v dithiothreitol (DTT) in serum-free media at pH 6-7;
ii) 5% PEG, 1% polyoxyethylene-polyoxypropylene block copolymer, and 0.31% w/v DTT in Hank's balanced salt solution (HBSS) at pH 6-7;
iii) 5% PEG, 1% polyoxyethylene-polyoxypropylene block copolymer, 0.8% medium viscosity G-groups alginate (MVG) and 0.31% w/v DTT in HBSS without Ca 2+ and Mg 2+ at pH 6-7; or
iv) 5% PEG, 0.8% medium viscosity G-groups alginate and 0.31% w/v DTT in HBSS without Ca 2+ and Mg 2+ at pH 6-7; and
b) the oil phase comprises polypropylene glycol (PPG) with 10% sorbitan oleate, wherein said oil phase further optionally comprises 0.02% or 0.2% triethanolamine.
6. The method of claim 1 , wherein the coating method has at least one of the following properties:
a) the ratio of the oil phase velocity to the water phase velocity is 350;
b) the ratio of the oil phase viscosity to the water phase viscosity is 3.5, 130, or 13;
c) the water phase is injected into the oil phase first at 50 μl/min and then reduced to 10 μl/min, while the oil phase is maintained at 3.5 ml/min; and
d) air is injected into the oil phase before injection of the water phase.
7. The method of claim 1 , wherein the step of purifying the conformally coated biomaterial and biomaterial-free coating material from the oil phase comprises the steps of:
a) centrifuging the coating device outflow to separate the oil phase from the conformally coated biomaterial and biomaterial-free coating material; and
b) optionally performing hexane extraction until the oil phase is completely eliminated from the conformally coated biomaterial and biomaterial-free coating material.
8. The method of claim 1 , wherein the step of separating the conformally coated biomaterial from the biomaterial-free coating material is performed by gradient centrifugation or by settlement of the conformally coated biomaterial by gravity.
9. The method of claim 1 , wherein:
a) said water phase comprises 5% PEG, 0.8% medium viscosity G-groups alginate (MVG), 75,000 islet cells/ml, and 0.31% w/v DTT in HBSS without Ca 2+ and Mg 2+ at pH 6-7;
b) said oil phase comprises PPG with 10% sorbitan oleate, wherein said oil phase optionally comprises 0.02% or 0.2% triethanolamine; and
c) said coating device comprises a flow chamber comprising a flow focusing region with a channel whose inner diameter is reduced from 10 mm to 1 mm with a focusing angle of 60 degrees, and a channel downstream of the flow focusing region that is about 1 mm in diameter.
10. The method of claim 1 , wherein:
a) said coating method comprises the steps of:
i) applying the oil phase to the flow chamber;
ii) optionally injecting air into the flow chamber through a catheter whose tip is localized 0.5 mm upstream of the base of the focusing region; and
iii) injecting the air, if present, and the water phase into the flow chamber through said catheter, wherein the water phase is first injected at 50 μl/min and then reduced to 10 μl/min, while the oil phase is maintained at 3.5 ml/min, such that the surface tension between the water and the oil phase causes the water jet to break up into microliter droplets comprising the conformally coated biomaterial and biomaterial-free coating material; and
b) said coating method optionally further comprises the steps of:
iv) collecting the outflow from the flow chamber;
v) centrifuging the outflow to separate the conformally coated biomaterial and biomaterial-free coating material from the oil phase;
vi) removing the oil phase supernatant from the conformally coated biomaterial and biomaterial-free coating material;
vii) resuspending the conformally coated biomaterial and biomaterial-free coating material in a composition comprising hexane;
viii) centrifuging the mixture of step vii) to separate the conformally coated biomaterial and biomaterial-free coating material from the hexane;
ix) removing the hexane supernatant;
x) resuspending the conformally coated biomaterial and biomaterial-free coating material in a composition comprising hexane and a buffer which does not comprise Ca 2+ and Mg 2+ ;
xi) centrifuging the mixture of step x) to separate the conformally coated biomaterial and biomaterial-free coating material from the hexane and a buffer which does not comprise Ca 2+ and Mg 2+ ;
xii) removing the hexane/buffer supernatant; and
xiii) resuspending the conformally coated biomaterial and biomaterial-free coating material in buffer containing Ca 2+ and Mg 2+ ; and
c) said coating method optionally further comprises the steps of:
xiv) layering solutions to form a density gradient capable of separating the conformally coated biomaterial and the biomaterial-free coating material;
xv) applying the conformally coated biomaterial and biomaterial-free coating material to the density gradient;
xvi) centrifuging the density gradient to separate the conformally coated biomaterial from the biomaterial-free coating material; and
xvii) removing the supernatant containing the biomaterial-free coating material.
11. The method of claim 1 , wherein the coating method has at least one of the following properties:
a) greater than 95% of the conformally coated biomaterial is purified from the biomaterial-free coating material;
b) the conformal coating around the biomaterial ranges from 10-20 μm in thickness; and
c) greater than 90% of the biomaterial introduced into the coating device is conformally coated.