Materials and methods for expansion of stem cells

Abstract

The subject invention concerns novel and translatable materials and methods for expansion of stem cells, such as mesenchymal stem cells (MSC), that significantly improve translational success of the cells in the treatment of various conditions, such as stroke. The subject invention utilizes cell self-aggregation as a non-genetic means to enhance their therapeutic potency in a microcarrier bioreactor. The subject invention integrates a cell aggregation process in a scalable bioreactor system. In one embodiment of the method, thermally responsive microcarriers (TRMs) are utilized in conjunction with a bioreactor system. Cells are cultured in a container or vessel in the presence of the TRMs wherein cells adhere to the surface of the TRMs. Once cells are adhered to the TRMs they can be cultured at a suitable temperature for cell growth and expansion, e.g., at about 37° C. After a period of time sufficient for cell growth and expansion on the TRMs, the cell culture temperature is reduced so that the cells detach from the TRMs. The detached cells are allowed to form cell clusters that are then cultured under conditions such that the clusters aggregate to form 3D aggregates. The 3D aggregates can be collected and treated to dissociate the cells (e.g., using enzymatic treatment, such as trypsinization). Dissociated cells can then be used for transplantation in methods of treatment or for in vitro characterization and study.

1 . A method for expanding a stem cell, wherein said method comprises culturing stem cells in a bioreactor system in the presence of a thermally responsive microcarrier (TRM), wherein stem cells adhere to the surface of said TRM; growing the adhered stem cells for a sufficient period of time for the stem cells to increase in numbers; detaching the stem cells from the TRM by reducing the culture temperature to a critical solution temperature that results in said adhered cells detaching from the surface of said TRM; providing said detached cells sufficient time to aggregate and form three-dimensional (3D) stem cell aggregates, wherein said 3D stem cell aggregates exhibit improved therapeutic potency. 
     
     
         2 . The method according to  claim 1 , wherein said bioreactor system comprises a spinner flask bioreactor or a rocking platform bioreactor, such as the WAVE Bioreactor. 
     
     
         3 . The method according to  claim 1 , wherein said culture conditions provide for rocking and/or agitation of said cells. 
     
     
         4 . The method according to  claim 1 , wherein said TRM is a microcarrier bead coated with or comprising a thermally responsive material selected from one or more of polyN-isopropylacrylamide (PNIPAAm), poly(allylamine hydrochloride)-co-poly(N-isopropylacrylamide), or poly(styrene sulfonate)-co-poly(N-isopropylacrylamide). 
     
     
         5 . The method according to  claim 1 , wherein said TRM comprises one or more of glass, polystyrene, poly(carprolactone), nylon, poly(ethylene terephthalate) (PET), gelatin, or dextran. 
     
     
         6 . The method according to  claim 4 , wherein said TRM optionally comprises a terminal coating of a layer of positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS), or serum, such as fetal bovine serum (FBS). 
     
     
         7 . The method according to  claim 1 , wherein said TRM has a diameter of between about 50 μm to about 500 μm; or from about 100 μm to about 200 μm. 
     
     
         8 . The method according to  claim 1 , wherein said stem cells are cultured in said bioreactor under hypoxic or low oxygen conditions. 
     
     
         9 . The method according to  claim 8 , wherein said hypoxic or low oxygen conditions comprise O 2  tension at between about 1% and about 10%; or between about 1% and about 3%. 
     
     
         10 . (canceled) 
     
     
         11 . The method according to  claim 1 , wherein said method further comprises collecting said 3D aggregates and treating said 3D aggregates to dissociate said aggregates into individual cells. 
     
     
         12 . The method according to  claim 11 , wherein said treating step comprises using an enzymatic agent. 
     
     
         13 . The method according to  claim 12 , wherein said enzymatic agent is trypsin. 
     
     
         14 . The method according to  claim 1 , wherein said stem cells are cultured in a container or vessel that comprises a surface or coating, wherein said surface or coating inhibits or prevents attachment of said stem cells to said container or vessel. 
     
     
         15 . The method according to  claim 1 , wherein the cells from said 3D cell aggregates exhibit one or more of the following: upregulated CXCR-4 expression; migration towards SDF-1; increased resistance to ischemic conditions; and/or enhanced expression of one or more anti-inflammatory cytokines and/or growth factors, such as IL-10, HGF, stanniocalcin 1 (STC-1), PGE-2, and/or IL-6. 
     
     
         16 . The method according to  claim 11 , wherein said cells are transplanted into a person or animal in need of treatment. 
     
     
         17 . (canceled) 
     
     
         18 . The method according to  claim 1 , wherein said stem cells are MSC from bone marrow. 
     
     
         19 . The method according to  claim 1 , wherein said stem cells are mammalian or human stem cells, or wherein said stem cells are mesenchymal stein cells (MSC). 
     
     
         20 . (canceled) 
     
     
         21 . A stem cell prepared according to a method that comprises culturing stem cells in a bioreactor system in the presence of a thermally responsive microcarrier (TRM), wherein stem cells adhere to the surface of said TRM; growing the adhered stem cells for a sufficient period of time for the stem cells to increase in numbers; detaching the stem cells from the TRM by reducing the culture temperature to a critical solution temperature that results in said adhered cells detaching from the surface of said TRM; providing said detached cells sufficient time to aggregate and form three-dimensional (3D) stem cell aggregates, wherein said 3D stem cell aggregates exhibit improved therapeutic potency; or
 a kit or article of manufacture comprising one or more containers and comprising one or more of said stem cells.   
     
     
         22 . The stem cell according to  claim 21 , wherein said stem cell is a human or mammalian cell. 
     
     
         23 . The stem cell according to  claim 21 , wherein said stem cell is a mesenchymal stem cell (MSC). 
     
     
         24 . The stem cell according to  claim 21 , wherein said stem cell is MSC from bone marrow. 
     
     
         25 . The stem cell according to  claim 21 , wherein said stem cell is a human mesenchymal stem cell (hMSC). 
     
     
         26 . The stem cell according to  claim 21 , wherein said stem cell is provided in a cell culture or cell storage medium, or wherein said stem cell is provided in a vessel or container. 
     
     
         27 . (canceled) 
     
     
         28 . A thermally responsive microcarrier (TRM), wherein said TRM is coated with or comprises a thermally responsive material that allows for cell adhesion at a first temperature but that provides for cell detachment at a second temperature, wherein said second temperature is less than said first temperature; or
 a bioreactor system comprising a cell culture vessel or container that contains said TRM, and optionally a cell culture fluid or medium.   
     
     
         29 - 41 . (canceled) 
     
     
         42 . A method for treating a disease or condition amenable to treatment with a stem cell, wherein the method comprises administering to a person or animal in need of treatment an effective amount of stem cells prepared according to a method that comprises culturing stem cells in a bioreactor system in the presence of a thermally responsive microcarrier (TRM), wherein stem cells adhere to the surface of said TRM; growing the adhered stem cells for a sufficient period of time for the stem cells to increase in numbers: detaching the stem cells from the TRM by reducing the culture temperature to a critical solution temperature that results in said adhered cells detaching from the surface a said TRM; providing said detached cells sufficient time to aggregate and form three-dimensional (3D) stem cell aggregates, wherein said 3D stem cell aggregates exhibit improved therapeutic potency. 
     
     
         43 . The method according to  claim 42 , wherein said stem cell is a human or mammalian stem cell, or wherein said stem cell is a mesenchymal stem cell. 
     
     
         44 - 49 . (canceled) 
     
     
         50 . A method for increasing or enhancing aggregation of stem cells during in vitro cell culture, the method comprising culturing said stem cells under conditions or in the presence of one or more compounds that increases or enhances actin-mediated contractility or polymerization in said stem cells. 
     
     
         51 . The method according to  claim 50 , wherein said stem cell is a human or mammalian cell. 
     
     
         52 . The method according to  claim 50 , wherein said stem cell is a mesenchymal stem cell. 
     
     
         53 . The method according to  claim 50 , wherein said stem cell is a human mesenchymal stem cell. 
     
     
         54 . The method according to  claim 50 , wherein said compound is selected from insulin, ecdysterone, ATP, fesselin, surfactant proteins A and D, cortactin, sphingosine-1-phosphate (S1P), or j asplakinolide (JASP).