JPWO2018183199A5 - - Google Patents

Claims (49)

a) Temperature-sensitive cell-stabilizing biomaterials, and
b) Bioactive renal cell (BRC) population,
It is an injectable preparation containing
The temperature-sensitive cell-stabilizing biomaterial is hydrogel.
(i) At about 8 ° C. or lower, the substantially solid state is maintained, and the substantially solid state is a gel state.
(ii) remain substantially liquid at near or above outside air temperature, and (iii) from solid to liquid between approximately 8 ° C and near or above outside air temperature. A hydrogel with a transition state,
The hydrogel contains extracellular matrix proteins of recombinant origin, is derived from the extracellular matrix obtained from the kidney or another tissue or organ, or contains gelatin.
The injectable product. The injectable preparation according to claim 1, wherein the gelatin is derived from type I αI collagen. BRC populations are coated with a biomaterial, deposited on the biomaterial, embedded in the biomaterial, attached to the biomaterial, seeded in the biomaterial, or captured in the biomaterial, claimed The injectable preparation according to Item 1. The injectable formulation of claim 1, wherein the biomaterial is composed of a porous foam, gel, liquid, beads, or solid. The injectable preparation according to claim 2, wherein the gelatin is derived from porcine type I αI collagen or recombinant human type I αI collagen. The injectable formulation according to claim 1, wherein the BRC is a selected renal cell (SRC) population. The BRC or SRC population contains a high proportion of one or more cell populations compared to the starting renal cell population and lacks or lacks the other one or more cell populations. , The injectable formulation according to claim 6. The injectable formulation of claim 7, wherein the BRC or SRC population is enriched for tubular renal cells. The injectable formulation of claim 8, wherein the BRC or SRC population exhibits a cellular morphology suggestive of tubular renal cells. The injectable formulation of claim 8, wherein the BRC or SRC population is characterized by phenotypic expression of one or more tubular epithelial cell markers. The injectable formulation of claim 10, wherein the tubular epithelial cell marker comprises CK18 and / or GGT1. The injectable formulation of claim 8, wherein the BRC or SRC population exhibits a cell proliferation rate suggestive of live metabolically active renal cells. 12. The injectable formulation of claim 12, wherein the BRC or SRC population is characterized by phenotypic expression of one or more viability markers and / or functional markers. 13. The injectable formulation of claim 13, wherein the one or more viability markers and / or functional markers comprise VEGF and / or KIM-I. The injectable formulation according to claim 12, wherein the BRC or SRC population is characterized by LAP and / or GGT enzyme activity. The injectable preparation according to claim 1, wherein gelatin is contained in the preparation at a ratio of about 0.5% to about 1% (w / v). The injectable preparation according to claim 1, wherein gelatin is contained in the preparation at a ratio of about 0.8% to about 0.9% (w / v). The injectable preparation according to claim 1, further comprising a cell survival agent. Cell survival agents include antioxidants, oxygen carriers, growth factors, cell stabilizing factors, immunomodulators, cell mobilizing factors, cell adhesion factors, anti-inflammatory agents, immunosuppressants, angiogenic factors, and wound healing factors. The injectable formulation of claim 18, comprising an agent selected from the group consisting of. The injectable preparation according to claim 18, wherein the cell viable agent is selected from the group consisting of human plasma, human platelet lysate, fetal bovine plasma, or bovine pituitary extract. a) Decellularized kidneys of human or animal origin, or cell-stabilized biomaterials structurally made by three-dimensional bioprinting, and
b) Bioactive renal cell (BRC) population,
A implantable formulation, including. The preparation according to any one of claims 1 to 21, further comprising a product secreted by a renal cell population. a) A biomaterial containing approximately 0.88% (w / v) gelatin, wherein the gelatin is derived from type I αI collagen, and the biomaterial.
b) A composition comprising an SRC population, wherein the SRC population comprises an enriched population of tubular renal cells and has a density greater than about 1.04 g / mL.
Injectable formulation containing. A method for preparing an injectable formulation containing a temperature sensitive cell stabilizing biomaterial and a bioactive renal cell mixture.
i) Steps to collect cortical tissue from donor / recipient;
ii) Isolation of renal cells from renal tissue by enzymatic digestion and proliferation of renal cells by standard cell culture techniques;
iii) The step of subjecting the collected renal cells to isolation beyond a density boundary or density interface or a single-step discontinuous gradient to obtain an SRC population;
iv) Containing steps to reconstitute bioactive cells using a gelatin-based hydrogel biomaterial, the gelatin is derived from type I αI collagen.
The method. 24. The method of claim 24, wherein the selected renal cells contain an enriched population of tubular renal cells and have a density greater than about 1.04 g / ml. 24. The 24. Method. 24. The method of claim 24, wherein the renal cells are enriched for tubular renal cells. 24. The method of claim 24, further comprising monitoring the cell morphology of renal cells during cell proliferation. 28. The method of claim 28, wherein the renal cells exhibit a cellular morphology suggestive of tubular renal cells. 24. The method of claim 24, further comprising monitoring the cell proliferation rate of renal cells for each cell passage. 30. The method of claim 30, further comprising monitoring renal cell count and viability with reagents for assessing metabolic activity. 24. The method of claim 24, further comprising monitoring renal cells for phenotypic expression of one or more viability markers and / or functional markers. 32. The method of claim 32, wherein the one or more viability markers and / or functional markers comprise VEGF and / or KIM-1. 24. The method of claim 24, further comprising monitoring renal cells for phenotypic expression of one or more tubular epithelial cell markers. 34. The method of claim 34, wherein the tubular epithelial cell marker comprises CK18 and / or GGT1. 24. The method of claim 24, further comprising monitoring renal cell function by gene expression profiling or measurement of enzyme activity. 36. The method of claim 36, wherein the enzyme activity measured is the activity of LAP and / or GGT. 24. The method of claim 24, wherein the kidney cells are obtained from an autologous or allogeneic kidney sample. 24. The method of claim 24, wherein the kidney cells are obtained from a non-autologous kidney sample. 38. The method of claim 38, wherein the sample is obtained by renal biopsy. 24. The method of claim 24, wherein the SRC is resuspended in a liquefied gelatin solution at 26-30 ° C. 41. The method of claim 41, wherein the SRC is resuspended in a gelatin solution sufficient to achieve an SRC concentration of 100x106 cells / ml. 24. The method of claim 24, further comprising quenching the SRC / gelatin solution to stabilize the biomaterial so that the SRC remains suspended in the gel during storage. 43. The method of claim 43, wherein the formulation is stored in a temperature range of 2-8 ° C. 42. The method of claim 42, further comprising the addition of a cell viable agent. Cell survival agents include antioxidants, oxygen carriers, growth factors, cell stabilizing factors, immunomodulators, cell mobilizing factors, cell adhesion factors, anti-inflammatory agents, immunosuppressive agents, angiogenic factors, and wound healing factors. 45. The method of claim 45, comprising an agent selected from the group consisting of. 45. The method of claim 45, wherein the cell viable agent is selected from a group consisting of human plasma, human platelet lysates, fetal bovine plasma, or bovine pituitary extract. The injectable pharmaceutical product according to any one of claims 1 to 20 and 23 for treating kidney disease in a subject, which is injected through a 18 to 30 gauge needle. The injectable formulation according to claim 48, wherein the diameter of the needle is about 27 gauge, about 26 gauge, about 25 gauge, about 24 gauge, about 23 gauge, about 22 gauge, about 21 gauge, or about 20 gauge.