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US-12623003-B2 - Three-dimensional microporous scaffold device for cell culture

US12623003B2US 12623003 B2US12623003 B2US 12623003B2US-12623003-B2

Abstract

An in vitro method of preparing insulin-producing cell clusters for transplantation into a subject, comprising (a) seeding pancreatic progenitor cells onto a three-dimensional, porous scaffold at a seeding density greater than about 12.5 million cells per cm 3 of scaffold and less than about 150 million cells per cm 3 of scaffold, wherein the scaffold comprises a plurality of pores having an average pore diameter greater than about 225 μm and less than about 550 pm, and (b) culturing the cells on the scaffold for more than about 3 days in culture medium to obtain insulin-producing cell clusters within the pores of the scaffold for transplantation into a subject. In exemplary aspects, the pancreatic progenitor cells are cells derived from pluripotent stem cells.

Inventors

  • Lonnie D. Shea
  • Richard Youngblood
  • Tadas Kasputis

Assignees

  • THE REGENTS OF THE UNIVERSITY OF MICHIGAN

Dates

Publication Date
20260512
Application Date
20190521

Claims (14)

  1. 1 . An in vitro method of preparing insulin-producing cell clusters, comprising: a) seeding pancreatic progenitor cells onto a three-dimensional, porous scaffold comprising a synthetic polymer at a seeding density greater than 12.5 million cells per cm 3 of scaffold and less than 250 million cells per cm 3 scaffold, wherein the scaffold comprises a plurality of pores having an average pore diameter of 250 μm to 425 μm; and b) culturing and differentiating the cells on the scaffold in vitro for at least 4 days to obtain insulin-producing cell clusters, wherein at least a portion of each insulin-producing cell cluster is in a pore of the scaffold.
  2. 2 . The method of claim 1 , wherein the seeding density is greater than 25 million cells per cm 3 scaffold and less than 125 million cells per cm 3 of scaffold.
  3. 3 . The method of claim 1 , wherein step (a) comprises seeding a volume of a solution comprising the pancreatic progenitor cells onto the three-dimensional, porous scaffold, wherein the volume is not more than 30 μL.
  4. 4 . The method of claim 1 , wherein the pancreatic progenitor cells are Stage 4 pancreatic progenitor cells expressing PDX1 and NKX6.1, optionally, wherein the Stage 4 pancreatic progenitor cells expressing PDX1 and NKX6.1 are derived from pluripotent stem cells.
  5. 5 . The method of claim 4 , wherein the pluripotent stem cells are human pluripotent stem cells (hPSCs).
  6. 6 . The method of claim 1 , wherein, prior to step (a), the method comprises treating the pancreatic progenitor cells with a cell dissociation agent.
  7. 7 . The method of claim 1 , wherein step (b) comprises culturing the cells on the scaffold in vitro for 4 days to 24 days to obtain insulin-producing cell clusters.
  8. 8 . The method of claim 1 , wherein the cells seeded onto the scaffold express and secrete extracellular matrix (ECM) proteins within the scaffold, wherein the ECM proteins comprise one or more of collagen IV, laminin and fibronectin.
  9. 9 . The method of claim 1 , wherein the scaffold does not contain and is not coated with collagen, laminin, or fibronectin prior to the seeding step (a).
  10. 10 . The method claim 1 , wherein the scaffold is fabricated with salt porogens having an average diameter of 250 μm to 425 μm.
  11. 11 . The method of claim 1 , wherein the scaffold comprises polyethylene glycol.
  12. 12 . The method of claim 1 , wherein the synthetic polymer comprises poly(lactide-co-glycolide) (PLG) and the average pore diameter is about 370 μm±37 μm.
  13. 13 . The method of claim 1 , wherein the synthetic polymer comprises poly(ethylene glycol) (PEG), poly(lactide-co-glycolide) (PLG), or a combination thereof.
  14. 14 . A method of treating a patient with an insulin deficiency, the method comprising: a. seeding pancreatic progenitor cells onto a three-dimensional, porous scaffold comprising a synthetic polymer at a seeding density greater than 12.5 million cells per cm 3 of scaffold and less than 250 million cells per cm 3 of scaffold, wherein the scaffold comprises a plurality of pores having an average pore diameter of 250 μm to 425 μm; b. culturing and differentiating the pancreatic progenitor cells on the scaffold in vitro for at least 4 days into insulin-producing cell clusters, wherein at least a portion of each insulin-producing cell cluster is in a pore of the scaffold; and c. administering the scaffold comprising the insulin-producing cell clusters to the patient with the insulin deficiency.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application is a U.S. National Phase of International Application No. PCT/US2019/033420, filed May 21, 2019, which claims priority to U.S. Provisional Application No. 62/674,370, filed on May 21, 2018, the contents of which are incorporated herein by reference. STATEMENT OF GOVERNMENT INTEREST This invention was made with government support under CA186786, and CA231996 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 4000 byte ASCII (Text) file named “53128A_Seqlisting.txt”; created on May 20, 2019. FIELD OF THE INVENTION The present disclosure relates generally to the fields of synthetic scaffold engineering and cell culturing. More specifically, the present disclosure relates to 3-dimensional (3D) synthetic scaffolds and uses thereof for the improved growth and differentiation of stem cells. BACKGROUND Type I diabetes (T1D) is a chronic metabolic disorder characterized by autoimmune destruction of the pancreatic β-cells that results in the need for life-long insulin therapy. This disease represents 5-10% of the diagnosed cases of diabetes, corresponding to more than 1.25 million individuals in the United States [Centers for Disease Control and Prevention 2017 Diabetes Report Card, CDC. (2015) 16]. Several secondary metabolic disorders can arise from this disease, as well, such as retinopathy, neuropathy, nephropathy, stroke and heart failure [Daneman, Type 1 diabetes, Lancet Lond. Engl. 367 (2006) 847-858. doi:10.1016/S0140-6736(06)68341-4; Tiwari et al., Clin. Pharmacol. Biopharm. 3 (2014). doi:10.4172/2167-065X.1000117]. Although exogenous insulin injections have decreased mortality, hypoglycemic events and macrovascular complications persist [Pambianco et al., Diabetes. 55 (2006) 1463-9; Bittencourt et al., Atherosclerosis. 240 (2015) 400-401. doi:10.1016/j.atherosclerosis.2015.04.013; and Kalra et al., Indian J. Endocrinol. Metab. 17 (2013) 819-834. doi:10.4103/2230-8210.117219]. Thus, recent research has turned to cell-based therapies focused on replacing lost insulin-producing cells. Enthusiasm in cell replacement therapies for diabetes was driven, in part, by the progress in allogeneic islet transplantation with the Edmonton protocol [Ryan et al., Diabetes. 54 (2005) 2060-9; Shapiro et al., N. Engl. J. Med. 355 (2006) 1318-1330. doi:10.1056/NEJMoa061267; Hering et al., Diabetes Care. 39 (2016) 1230-1240. doi:10.2337/dc15-1988; Brennan et al., Long-Term Follow-Up of the Edmonton Protocol of Islet Transplantation in the United States, Am. J. Transplant. Off. J. Am. Soc. Transplant. Am. Soc. Transpl. Surg. 16 (2016) 509-517. doi:10.1111/ajt.13458, O'Connell et al., Australian Islet Transplant Consortium, Multicenter Australian trial of islet transplantation: improving accessibility and outcomes, Am. J. Transplant. Off. J. Am. Soc. Transplant. Am. Soc. Transpl. Surg. 13 (2013) 1850-1858. doi:10.1111/ajt.12250]. Recently, promising results from a European consortium of islet transplant centers showed excellent glycemic control and absence of hypoglycemia reported in approximately 80% of patients at 1 year and 60% at 5 years [Lablanche et al., Diabetes Care. 38 (2015) 1714-1722. doi:10.2337/dc15-0094]. However, the widespread application of islet transplantation has been tempered by the lack of availability of islets and the need for life-long immunosuppression [Stegall et al., Transplantation. 61 (1996) 1272-1274; Shapiro et al., Diabetes. 62 (2013) 1377-1378. doi:10.2337/db13-0019]. The lack of available islets has led to the investigation of human pluripotent stem cells (hPSCs) as an unlimited source of functional β-cells. Initial findings from the Kieffer and Baetge/D'Amour groups demonstrated the production of pancreatic progenitors and, subsequently, insulin-producing β-like cells in vitro. In the Kieffer lab, these cells could further differentiate following transplantation to normalize blood glucose levels after approximately 3-4 months [Rezania et al., Diabetes. 61 (2012) 2016-2029. doi:10.2337/db11-1711; Kroon et al., Nat. Biotechnol. 26 (2008) 443-452. doi:10.1038/nbt1393]. More recently, in vitro culture protocols have developed hPSC-derived β-cells that induce normoglycemia over shorter times after transplantation [Velazco et al., Stem Cell Rep. (2019). doi:10.1016/j.stemcr.2018.12.012; Rezania et al., Nat. Biotechnol. 32 (2014) 1121-1133. doi:10.1038/nbt.3033; Pagliuca et al., Cell. 159 (2014) 428-439. doi:10.1016/j.cell.2014.09.040; Pepper et al., Diabetes. (2018). doi:10.2337/db18-0788]. Additionally, suspension cultures utilized for aggregated hPSC-derived β-cell production provide procedures that are scalable to generate sufficient glucose-responsive cells [Velazco, 2019,