METHOD OF MANUFACTURING PANCREAS ISLET OF LANGERHANS MIMICS USING INDUCED PLURIPOTENT HUMAN STEM CELLS

Abstract

The present disclosure provides a method of manufacturing pancreas Islet of Langerhans (IOL) mimetics using induced human pluripotent stem cells (iPSc) and porous micro carrier scaffolds that allow for subsequent vascularization and/or innervation.

Claims

1. A method of manufacturing Islet of Langerhans (IOL) pancreas mimics, comprising: culturing in liquid media pancreatic cells or pancreatic progenitor cells and biocompatible microporous particles formed of a biodegradable material and having pores, under conditions that allow for coating of the cells into and on the particles and the pores and optionally differentiation of the progenitor cells in and on the particles and the pores; and degrading the particles under conditions that yield viable pancreatic cells having biological activity, thereby providing for a IOL mimic having a three dimensional population of the pancreatic cells or differentiated progenitor cells interspersed with microchannels.

2. The method of claim 1 wherein the culturing occurs in a microgravity reactor.

3. The method of claim 1 wherein the cells that are cultured are pancreatic progenitor cells and the conditions optionally include factors that provide for differentiation to alpha cells, beta cells, delta cells, or any combination thereof.

4. The method of claim 1 wherein the cells are obtained from iPSc.

5. The method of claim 1 wherein the particles are degradable by one or more enzymes.

6. The method of claim 1 wherein the particles comprise gelatin.

7. The method of claim 1 wherein the particles are degradable by trypsin, cellulase, dextranase, gelatinase, pepsin, pancreatin, papain, or bromelain.

8. The method of claim 1 wherein the particles have an average diameter of about 100 microns to about 250 microns.

9. The method of claim 1 wherein the particles have an average diameter of about 50 microns to about 200 microns.

10. The method of claim 1 wherein the pores have an average diameter of about 5 microns to about 40 microns.

11. The method of claim 1 wherein the pores have an average diameter of about 10 microns to about 30 microns.

12. The method of claim 1 further comprising encapsulating the IOL mimic having the cells and microchannels in a biocompatible polymer.

13. The method of claim 1 wherein about 1×10.sup.6 to about 5×10.sup.6 microparticles per liter of media are cultured with the cells.

14. The method of claim 1 wherein about 20×10.sup.6 to about 100×10.sup.6 cells per liter of media are cultured with the microparticles.

15. An implantable device comprising an IOL mimic having the cells and microchannels prepared by the method of claim 1.

16. The device of claim 15 wherein the cells comprise a three dimensional population of cells having a diameter of about 10 microns to about 1 millimeter.

17. The device of claim 15 wherein the cells comprise a three dimensional population of having a diameter of about 100 microns to about 200 microns.

18. The device of claim 15 wherein the IOL is encapsulated in a film having a thickness of about 100 microns to about 200 microns.

19. The device of claim 15 which has about 5×10.sup.6 to 15×10.sup.6 cells.

20. A method to inhibit or treat diabetes, comprising: administering to a patient in need thereof the device of claim 15.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0016] FIG. 1 shows a cross sectional view of a pancreas Islet of Langerhans (IOL) structure. The pancreas has two functions: to make hormones (endocrine) and to produce enzymes for digestion (exocrine). The islets of Langerhans make the hormones insulin and glucagon. The alpha cells 10 make the glucagon and the beta cells 20 produce insulin and the delta cells 30 make somatostatin.

DETAILED DESCRIPTION

Bioreactors

[0017] The use of bioreactors to produce various human cell and stem cell microstructures has been described.

[0018] For example, WO 97/16536, which is incorporated by reference herein, refers to methods for the ex vivo proliferation and differentiation of neonatal and/or adult human or non-human pancreatic islets. The cells are cultured in a microgravity environment and an aggregation medium is employed. The intention is to produce products useful for the treatment of diabetes. It is important to note that WO 97/16536 describes work with adult human tissue and not embryonic stem cells. The tissue used is a donated pancreatic cell from cadavers. The work involves disaggregating the pancreatic islets and then reassembling them using a culture system, and there is a proposal to co-culture with other adult cell types.

[0019] In addition, US 2011/0027880, which is incorporated by reference herein, describes the use of a microgravity reactor to produce pancreatic islands. Three-dimensional (3D) insulin-producing cell clusters derived from stem cells (human embryonic stem cells) are provided by the present disclosure, together with a method for their production using a microgravity bioreactor cell culture system.

Exemplar Methods Using Stem Cells and Microporous Carriers

[0020] The present disclosure provides for the use of stem cells including iPSc; a microgravity reactor and porous micro carrier scaffolds that are subsequently enzymatically digested for the production of Islets of Langerhans (IOL) mimics that can be subsequently vascularized when implanted into a human patient. FIG. 1 shows a diagram of a cross section containing alpha, beta, and delta cells.

[0021] Human pluripotent stem cells (hPSC) have the ability to self renew and proliferate and to differentiate into a wide range of cell types, including pancreas islet cells. Although a number of methods for direct conversion of one differentiated cell type to another have been described, the majority of successful strategies for generating large numbers of differentiated cells attempt to recapitulate the normal development progression to the terminally differentiated state. In these strategies, pluripotent cells are progressively directed by external signals, first to one of the three germ lineages, and then to more specific downstream cell types. The pancreas is derived from the endoderm lineage, which also gives rise to the lung, liver, and gastrointestinal tract.

[0022] In one embodiment, inducers of a pathway are used to first generate pancreas progenitor cells, which are then attached to a micro carriers and incubated in a microgravity reactor for about 3 to about 25 days. The micro carriers are then harvested, exposed to an enzyme, such as trypsin, to degrade the micro carrier matrix, which in one embodiment comprises gelatin, to result in a porous IOL mimic that is then optionally stored in growth media until it is ready for implantation.

[0023] In one embodiment, the differentiation process starts with human skin cells that are reprogrammed, e.g., by transfection with Sendai virus, to induced pluripotent stem cells (iPSc) by techniques well known in the art. Once the iPSc are generated, the next step is to induce endoderm formation which is generally about a 3 to 7 day process induced by one or more of LY, ActivinA, BMP4, and FGF2 signaling factors. Next gut patterning occurs which again is about a 3 to 5 day process induced by, for example, IWR signaling factor. The next steps involve pancreas specification, proliferation, and progenitor formation that is about a 10 to 20 day process involving growth and signaling factors, such as BMP4, IWr1, SB, HGF, CHIR, vEGF, TGFb, CE, or any combination thereof.

[0024] For exemplary factors and conditions for pancreatic lineage differentiation from, for instance, human stem cells, see Shih et al., Ann. Rev. Cell Dev. Biol., 29:81 (2013) the disclosure of which is incorporated by reference herein

Microcarrier Beads

[0025] Macroporous beads, in which anchorage-dependent cells have the possibility to utilize the interior surface, substantially reduce the problems associated with the culture of these cells. Microcarriers have been manufactured from different synthetic materials including dextran, such as GE Biosciences Cytodex, polyacrylamide, polystyrene and cellulose. Cell attachment to these charged microcarriers are mediated by ionic attractions. Cells also attach to gelatin, but through a different mechanism: a protein, fibronectin, has a biospecific binding to gelatin and as the cells has an affinity to this protein they will attach to microcarriers of gelatin, which is susceptible to proteolytic enzymes. Cells may thus be released with almost 100% viability by dissolution of the matrix with an enzyme, e.g., trypsin. Table 1 shows the typical properties of exemplary micro carrier scaffolds that may be employed in the present methods.

TABLE-US-00001 TABLE 1 Particle diameter (μm) 130-180 130-180 Volume (mL/g dry) 12-18 10-16 Density (g/mL) 1.04 1.04 Microcarriers 1,000,000 800,000 number/gm Average Pore diameter 20 20 (μm)

[0026] Pancreas progenitor cells are cultured on the beads that are loaded into a microgravity reactor, see, e.g., U.S. Pat. Nos. 5,437,998, 5,155,035 and 5,989,913, the disclosures of which are incorporated by reference herein. Cultures of cells grown on CultiSpher (Percell Biolytia, Astorp Sweden), can be scaled-up in steps of 50 times; for instance, cells harvested from a 1 liter fermentor are sufficient to inoculate a 50 liter fermentor. This is possible due to the macroporous structure and, in one embodiment, a digestible matrix such as a digestible gelatin matrix. The macroporous structure allows the cells to increase from about 10 to 20 cells up to about 2000 to 3000 cells on each bead. As the matrix may be digested with tissue culture grade enzyme, e.g., trypsin, many of not all of these cells can be recovered and used for scale-up purposes.

[0027] Used at 1 g/L, cell concentrations of 10×10.sup.6 cells/mL can be obtained if an efficient oxygen supply system is used. If used in higher concentrations than 2 g/L in standard systems, cells only grow on the outer layer of the microcarriers. Growth is dependent on the oxygen gradient within the system. Cells only grow as long as they are provided with sufficient amounts of oxygen. The pH is usually controlled by carbon dioxide; if a large number of cells are growing inside the beads they will produce an acidic environment. Unless this is controlled it will affect both cell growth and product formation.

[0028] The invention will be described by the following non-limiting example.

Example

[0029] A 1 L microgravity reactor is inoculated. CultiSpheres is used at about 2 g/L and contains about 2×10.sup.6 beads. Each bead is inoculated with at least 10 cells. For example, about 20×10.sup.6 cells may be used for enhanced attachment and growth. If this concentration is about 0.1×10.sup.6 cells/mL and the minimum volume that can be used is about 0.5 L, about 50×10.sup.6 cells are used. The culture may thus be started with about 50×10.sup.6 cells, which will supply each CultiSphere with about 10 to about 20 cells up to about 2000 to about 3000 cells on each bead. To form the microstructured IOL mimic microchannels for subsequent vascularization, the microcarriers are evenly dispersed. Duplicate samples of about 0.5 mL are taken and after sedimentation of beads, 0.3 mL of the supernatant is removed. Add 0.8 mL trypsin (0.25% w/v in PBS). Mix and incubate at 37° C. for 20 minutes. If the microcarriers have not dissolved after this time, the trypsin concentration is increased. As the matrix is easily digested with tissue culture grade trypsin all the cells can be recovered and used for scale-up and implantation or therapeutic purposes. For example, a protease may be added when the cells have differentiated, e.g., from about 18 to 24 days of starting the culture, and/or insulin secretion is detected. In one embodiment, the protease is contacted with the cell/bead mixture for about 0.2 to 24 hours.

[0030] It is to be appreciated that using this process it would be possible to also manufacture other mammalian organ types such as liver, kidney, lung, spleen, brain, eye, skin, nerve, blood vessels, bladders, intestine and heart tissue all of which would benefit from having a porous structure that could be vascularized or innervated to form functional human organs if implanted into a body or used for drug screening and efficacy testing.

[0031] The subject matter herein is described by example and different ways of practicing the subject matter have been described. However the subject matter covered by this application is not limited to any one specific embodiment or use or their equivalents. While particular embodiments of the method for fabricating cell micro arrays with subsequent drug dosing have been described it will be appreciated by those skilled in the art that changes and modifications may be made thereto without departing from the invention and as set forth in the following claims.