Adaptation of hollow-fiber-based cell culture technology for manufacturing (1) neo-islets for the treatment of type 1 and type 2 diabetes mellitus, and (2) exosomes from various cell types for use in the treatment of different organ injuries and diseases

Abstract

To provide methods for generating exosomes (extracellular nanovesicles) and / or neo-islets derived from mesenchymal or adipose stem cells or islets or other cells in a hollow fiber cell expansion (HFCE) system.SOLUTION: The problem is solved by a method comprising expanding mesenchymal or adipose stem cells and islet cells in a hollow fiber cell expansion (HFCE) system, and collecting neo-islets from the HFCE system.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates generally to the fields of cell biology and medicine. In particular, the present disclosure relates to the production of cells and exosomes for use in treating a subject, and more particularly, to the production of cells and exosomes useful in the treatment of diabetes mellitus. [Background technology]

[0002] Mesenchymal stem or stromal cells (MSCs) from bone marrow, adipose, umbilical cord, placenta, amniotic fluid, and other sources are non-embryonic "adult" stem cells that possess potent anti-inflammatory, anti-apoptotic, mitogenic, angiogenic and angiogenic protective, immunomodulatory, antifibrotic, antithrombotic, and antibiotic paracrine activities that have been used in promising treatments of various acute and chronic diseases and organ injuries. Most specifically, we have shown (Westenfelder C, Gooch A. et al. Stem Cell Transduction Medicine Vol. 6, No. 7, 1631-1643, July 2017) that coaggregates of allogeneic MSCs and allogeneic culture-expanded pancreatic islet cells durably correct T1DM in a mouse model of autoimmune T1DM that closely resembles human and canine T1DM (NOD mice). The MSC component of NI provides robust protection against alloimmune and autoimmune attack of insulin-producing endocrine islet cells in vivo, obviating the use of potentially toxic anti-rejection drugs that can cause damage to transplanted islets in severe infections, malignancies, and liver and kidney disease. In addition, the NI technology allows for the generation of 80+ therapeutic doses from a single pancreas donor, in contrast to islet transplant protocols in which diabetic recipients require four or more donors for one single treatment. MSCs in culture release beneficial cytokines and growth factors that mediate their pleiotropic effects. In addition, like essentially all cells, MSCs release exosomes (30-100 nm in diameter) within their microenvironment, which are taken up by neighboring cells and then result in paracrine signaling within the target cells. Signaling occurs through the lateral transfer of mRNA, miRNA, proteins, and lipids, known to exert beneficial effects that are essentially identical to those of intact MSCs. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Westenfelder C, Gooch A. et al. Stem Cell Transition Medicine Vol. 6, No. 7, pp. 1631-1643, July 2017 Summary of the Invention

[0004] Described herein are a) mesenchymal and / or adipose stem cells, b) pancreatic islet cells, c) mesenchymal and / or adipose stem cell-derived exosomes, and d) exosomes derived from other cell types, such as endothelial, muscle, and other cultured cells. NeoIslets (NIs) are further described, which contain mesenchymal and / or adipose stem cells, and pancreatic islet cells.

[0005] Disclosed herein are methods for generating exosomes (extracellular nanovesicles). Such methods may include growing mesenchymal or adipose stem cells or pancreatic islets, or other cells in a hollow fiber based cell expansion (HFCE) system, and harvesting exosomes released by the culture-grown mesenchymal, adipose stem cells, or other cells from the HFCE system.

[0006] In an embodiment of this method, cells are grown to 70-95% confluence. In a further embodiment, the culture medium perfused through the HFCE system may be adjusted to enhance release of exosomes into the lumen of the hollow fibers.

[0007] Embodiments include exosomes produced by such methods, and methods of treating a subject suffering from T1DM, T2DM, or a related microvascular disease by treating the subject with exosomes produced by the above methods. In certain embodiments, the exosomes have been cryopreserved for at least two years prior to treatment.

[0008] Method embodiments for generating neo-islets are provided. Certain embodiments include growing mesenchymal or adipose stem cells and islet cells in an HFCE system and harvesting the neo-islets from the HFCE system. In embodiments of the method, the cells are grown to 70-95% confluence.

[0009] In embodiments, NeoIstel may comprise a) differentiated islet cells and mesenchymal or adipose stem cells, and / or redifferentiated islet cells and mesenchymal or adipose stem cells. In certain embodiments, the islet cells and stem cells are present in the neo-islets at an islet cell:stem cell ratio of about 1:100, 1:75, 1:50, 1:25, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 25:1, 50:1, 75:1, or 100:1.

[0010] In certain embodiments, the harvested neo-islets are placed and / or encapsulated in a hydrogel.

[0011] Embodiments include neo-islets produced by such methods.

[0012] Embodiments include methods of treating subjects suffering from T1DM or T2DM and associated microvascular disease with neo-islets produced by the above methods.

[0013] Also described are methods of treating a subject with T1DM or T2DM, comprising administering to the subject NIs with or without exosomes derived from MSCs or other cultured cells.

[0014] The NI technology disclosed herein impairs, where appropriate, the multiple functions of MSCs and exosomes required to repair and protect the damaged capillary system in the above diseases and disorders.

[0015] "Treating" or "treatment" does not require a complete cure. It means that the symptoms of the underlying disease are at least reduced, and / or one or more of the underlying cellular, physiological or biochemical causes or mechanisms are diminished and / or eliminated. Reduced, as used in this context, is understood to mean the state of the disease, as opposed to the physiological state of the disease.

[0016] As used herein, a "therapeutically effective amount" is an amount sufficient to act as a treatment as defined above, which can be determined, for example, by standard techniques used to monitor and / or diagnose a particular disease state.

[0017] As used herein, the terms "comprising," "including," "containing," "characterized by," and their grammatical equivalents do not exclude additional, unrecited elements or method steps, but also include the more restrictive terms "consisting of" and "consisting essentially of."

[0018] In conclusion, the aim of using the customized hollow fiber-based cell culture system is to enable the expansion and highly efficient production of MSCs, pancreatic islet cells, and therefore NIs for the treatment of diabetes. In addition, various cultured cell types in the hollow fiber-based cell culture system can be programmed to generate large numbers of exosomes that are therapeutically desirable, have enhanced intrinsic therapeutic qualities, and are used to treat specific diseases. It is also envisioned that the widespread use of the developed highly efficient hollow fiber-based cell culture technology will prove cost-saving for the entire healthcare system. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a diagram for explaining an embodiment of the present invention. [Diagram 2] FIG. 1 is a diagram for explaining an embodiment of the present invention. [Diagram 3] FIG. 1 is a diagram for explaining an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The illustrations presented in this disclosure are not intended to be actual drawings of any particular composition, but are merely presented to illustrate exemplary embodiments, and therefore the drawings are not necessarily drawn to scale.

[0021] In the present disclosure, the term mesenchymal stem cells includes mesenchymal and / or adipose stem cells, and b) includes mesenchymal and / or adipose stem cell-derived exosomes. Although certain exemplary embodiments have been described in conjunction with the drawings, those skilled in the art will recognize and understand that the scope of the present disclosure is not limited to the embodiments expressly shown and described in this disclosure. Rather, many additions, deletions, and modifications to the embodiments described in this disclosure may be made to produce embodiments that are within the scope of the present disclosure, such as those specifically claimed, including legal equivalents. In addition, features from one disclosed embodiment can be combined with features of another disclosed embodiment, as contemplated herein, and still fall within the scope of the present disclosure.

[0022] The present disclosure relates generally to the production of neoislets for the treatment of type 1 diabetes mellitus in companion animals such as dogs and human subjects. NeoIslets (NI) are coaggregates of culture-expanded pancreatic islet cells and cultured mesenchymal stem cells (MSCs) obtained from bone marrow or adipose tissue. The hollow fiber-based cell culture technology is a highly efficient and enclosed system, and therefore does not require clean rooms that are currently used for the growth of attached MSCs and other cells for both research and clinical use and suspension culture of various types of blood cells.

[0023] Therapeutic targets of the technology disclosed herein include type 1 and type 2 diabetes mellitus and their multi-organ complications (renal, ocular, cardiac, neurological and other), vascular ligation, autoimmune diseases, sepsis, acute and chronic kidney disease, cardiovascular and neurological diseases, solid organ transplantation, vascular rejection, wound healing, atherosclerosis, aging, and various degenerative retinal and neurological diseases. More specifically, the disclosed embodiments relate to the therapeutic use of mesenchymal stem cells from various sources as key components of NIs and their exosomes to repair and protect and stabilize diseased microvasculature, thereby improving outcomes and survival with T1DM in both dogs and humans.

[0024] It is envisioned that the hollow fiber cell culture (HFCE) system can be modified and used for the expansion of pancreatic islet cells, the generation of therapeutic doses of NIs, and the generation of exosomes from cultured MSCs and other cells programmed to confer specific therapeutic properties to the released exosomes for the treatment of various diseases such as acute renal failure, chronic kidney disease, stroke, heart attack, various autoimmune diseases, organ transplants, and others.

[0025] Use of HFCE systems to generate exosomes and NIs: (see paragraph above). Such HFCE systems are in some aspects similar in configuration to clinically used hemodialysis cartridges. These systems have the distinct advantage of offering a highly favorable surface area to culture medium ratio and flexibility regarding changes in culture conditions for both attached cells (e.g., mesenchymal stem, pancreatic islets, and other cell types) and cells in suspension culture, a technique used to produce neo-islets. In addition, various attached cell types grown in HFCE systems can be programmed to release exosomes with optimized therapeutic properties such as immunoisolation, anti-inflammatory, cytoprotection, angiogenesis, etc. Together, the efficacy and cost of cell culture, neo-islets, and exosome production in suitable HFCE systems are expected to be substantially better than traditional culture systems.

[0026] The production of exosomes (extracellular nanovesicles) is spontaneously released from MSCs, ASCs, and most other cell types when efficiently cultured in a suitable HFCE system (bioreactor) or flask system. They can be continuously harvested (surface marker expression, miRNA, RNA, DNA, peptides, cytokines, and lipid cargo), cryopreserved, or used fresh. The utilized cell culture medium in the HFCE system is the standard medium used by all experts in the field. However, modifications of the culture medium can be utilized to generate exosomes with desired therapeutic properties, such as extended anti-inflammatory, immunomodulatory, and trophic effects (anti-apoptotic, mitogenic, angiogenic, and others).

[0027] The production of NIs by the HFCE system described above utilizes culture-grown islet cells (insulin, glucagon, somatostatin, PPY producing cells and other non-endocrine cells) and culture-grown MSCs or ASCs. Suspension co-culture of islet cells and MSCs or ASCs in a suitably modified HFCE system results in efficient generation of NIs that can be used fresh for the treatment of T1DM, with appropriate characterization (associated gene expression profiles, glucose-sensitive insulin release assays, etc.), or cryopreserved for their later use.

[0028] With respect to the treatment of subjects with NIs, the contents of US patent application Ser. No. 15 / 261,750 are incorporated herein by reference in their entirety. Working Example

[0029] The following examples are provided for illustrative purposes only and are not to be construed as limiting the disclosure to the embodiments specifically disclosed therein.

[0030] Both type 1 and type 2 diabetes lead to progressively widespread end-organ damage, affecting the retina, coronary, nervous system, kidneys, and other organs through microvascular disease that damages the capillaries in these organs through endothelial and percutaneous acid dysfunction, microvascular obstruction and inflammation, vasoconstriction, vascular leakage, coagulation, and fibrosis. This microvascular damage also affects the capillaries of the islets of Langerhans in the pancreas. This pathological mechanism, which we hypothesize, can accelerate the destruction of insulin-producing cells, thereby hastening the complete loss of endogenous insulin production, i.e., greatly worsening the diabetic condition in patients. Therefore, we tested whether parenteral administration of mesenchymal and / or adipose-derived exosomes to db / db mice with advanced diabetic disease would improve glycemic control. This was observed along with an improvement in diabetic kidney disease.

[0031] Various vasculitides, autoimmune diseases, sepsis, acute and chronic renal diseases, cardiovascular and neurological diseases, solid organ transplants, vascular rejection, wound healing, atherosclerosis, aging, and various degenerative retinal diseases hold the promise that treatment with mesenchymal and / or adipose stem cells or their exosomes alone, or in combination with their parental stem cells, will favorably impact outcomes in this large group of microvascular diseases.

[0032] A male subject with type 2 diabetes presents with chronic kidney disease. MSCs are isolated from the subject and expanded in culture. The expanded MSCs are administered in an amount sufficient to ameliorate the symptoms suffered by the subject, the symptoms being related to microvasculature problems.

[0033] Detailed Example of Using the HFCE System to Generate Exosomes and NIs:

[0034] It is envisioned that the HFCE system will be used to produce exosomes from cultured MSCs, ASCs, and other target cells, as shown in Figure 1. The target cells from which exosomes (MSCs or ASCs, other cells) or NIs (islet cells, MSCs or ASCs) are to be produced are loaded and suspended in the top port in a suitable culture medium (Figure 1). The defined culture medium perfuses the interior and exterior space of the hollow fibers containing the cells to be expanded or co-cultured (MSCs or ACSs with islet cells). When the desired degree of cell proliferation is reached in the HFCE system, the cells are separated and harvested at the bottom of the HFCE system. When the desired degree of cell clustering is reached, the NIs are harvested at the bottom of the HFCE system. Exosomes from the cultured cells are harvested without separating the cultured cells.

[0035] Treatment of diabetic (type 1) subjects with NI consists of administering a weight-based dose of this composition to the intraperitoneal space of such subjects using ultrasound guidance and local anesthesia. Exome-based treatment of subjects with various diseases (autoimmune, transplant-related complications, ischemic injury, vasculitis, liver disease, neurodegenerative, retinal, cardiac, renal, and other diseases) is based on the intravenous administration of a predetermined dose of functionally "customized" (see above) exosomes.

Claims

[Claim 1] 1. A method for generating neo-islets, comprising: Cultivating mesenchymal or adipose stem cells and pancreatic islet cells in a hollow fiber cell expansion (HFCE) system; and harvesting neo-islets from the HFCE system.

Images