Florida State University Research Foundation, Inc.

Teng Ma

Yan Li

Sebastien Sart

Xuegang Yuan

Ang-Chen Tsai

Junho Kim

The subject invention concerns materials and methods for culture of cell aggregates. The subject invention utilizes three-dimensional (3-D) inserts comprising micro-channels having selected dimensions. The inserts are provided in or on tissue culture plates that can be supported on a programmable rocking platform/station, thereby providing for a hydrodynamic environment that promotes 3-D aggregation of cells cultured on the plates. The supporting rocker is programmed to provide motion that generates hydrodynamic conditions that support 3-D cell aggregation and long-term culture. The subject invention also concerns an apparatus comprising a tissue culture vessel that comprises a 3-D insert of the present invention, and a programmable rocking platform/station that can provide motion to the vessel provided thereon, thereby generating hydrodynamic conditions and wave motion that support 3-D cell aggregation and cell culture. The subject invention also concerns methods for growing 3-D aggregates of cells.

Three-dimensional culture device and methods for dynamic culture of cell aggregates

The subject invention concerns materials and methods for expansion of stem cells, such as mesenchymal stem cells (MSC), that improve translational success of the cells in the treatment of various conditions. The subject invention utilizes cell self-aggregation as a non-genetic means to enhance their therapeutic potency in a microcarrier bioreactor. In one embodiment of the method cells are cultured in a container or vessel in the presence of thermally responsive microcarriers (TRMs) wherein cells adhere to the surface of the TRMs. After a period of time the cell culture temperature is reduced so that the cells detach from the TRMs. The detached cells are allowed to form 3D aggregates. The 3D aggregates can be collected and treated to dissociate the cells. Dissociated cells can then be used for transplantation in methods of treatment or for in vitro characterization and study.

Materials and methods for expansion of stem cells

Improved hybrid neurovascular spheroids and methods for making the same are provided. In some embodiments of a method for making a hybrid neurovascular spheroid, the method includes i) propagating cortical cells to form a cortical spheroid; ii) propagating endothelial cells to form an endothelial spheroid; iii) propagating mesenchymal stem cells to form a mesenchymal cell culture; and iv) combining the cortical spheroid, endothelial spheroid, and mesenchymal spheroid under conditions to form the hybrid neurovascular spheroid.

Cortical spheroids and methods of making the same

The subject invention pertains to acellular extracellular matrix (ECM) and methods for preparing it. In one embodiment, the ECM is prepared from 3-D aggregates of pluripotent stem cells or from embryoid bodies. In one embodiment, the ECM is in the form of acellular microspheres. The stem cells used in the present invention can be embryonic stem cells (ESC). In a specific embodiment, the stem cells or embryoid bodies are from human. The ECM of the invention can be mass produced using PSC aggregates or embryoid bodies in large scale bioreactors. The acellular ECM produced using the present invention can be used for injection into a person or animal to regenerate tissue and/or treat a disease or condition. A method of the invention comprises growing stem cells in suspension culture as aggregates, e.g., in a bioreactor. Acellular ECM is then prepared from the cell aggregates by decellularization. In a specific embodiment, a decellularization reagent comprising Triton X-100 is used, followed by treatment with a DNase.

Extracellular matrix derived from stem cells and methods for production

The subject invention concerns materials and methods for growing and expanding MSC while maintaining their undifferentiated phenotype, self-renewal ability, and/or multi-lineage potential. In one embodiment, a method of the invention comprises i) seeding freshly isolated MSC on a planar surface or a 3-D scaffold and growing the cells under physiological or low Otension for a period of time sufficient to support formation of 3-D ECM network; ii) decellularizing the planar surface or 3-D scaffold; and iii) reseeding the decellularized planar surface or 3-D scaffold with MSCs, whereby the reseeded MSCs can be grown on the scaffold and maintain an undifferentiated phenotype. In one embodiment, the 3-D scaffold comprises or is composed of PET. In one embodiment, the MSC are human MSC (hMSC).

Mesenchymal stem cells (MSC) expansion methods and materials

Porous HCG scaffolds are provided in a perfusion bioreactor. Cells are seeded in the HCG scaffolds and cell culture media is perfused through the bioreactor to allow for cell seeding and growth. The cell culture media is removed, the HCG-cell constructs washed, and then preserved in the bioreactor with a perfusion comprising cryopreservation fluid comprising one or more of DMSO, trehalose, glycerol, ethylene glycol, or serum. The HCG-cell constructs (or the perfusion chambers containing them) are then removed from the bioreactor and placed in a cryopreservant media and maintained at about &#x2212;80&#xb0; C. The frozen HCG-cell constructs (or the chambers containing them) can then be stored at a suitable cryogenic temperature until needed. When needed, frozen HCG-cell constructs can be removed from cold storage and thawed using suitable means.

Materials and methods for cryopreserved bone constructs

An apparatus and method for a modular cell culture bioreactor comprises a plurality of chambers for cell culture; at least one reservoir containing a cell support medium; a plurality of conduits fluidly connecting the at least one reservoir with the plurality of chambers; and at least one pump fluidly connected through the plurality of conduits with the at least one reservoir and with the plurality of chambers to pump cell support medium therethrough; wherein each individual chamber of the plurality of chambers includes at least one three-dimensional matrix comprising polyethylene terephthalate, a plurality of channels carrying the cell support medium and having the matrix positioned in fluid communication therebetween, and at least two openings into each the channel, wherein a first the opening is in fluid connection with the pump and a second the opening is in fluid connection with the reservoir.

Modular cell culture bioreactor

A bioreactor for cell culture includes a chamber having three channels therethrough for conducting fluids, the three channels including an upper channel, a middle channel, and a lower channel, each the channel having an inlet and an outlet approximately opposite along the channel from the inlet. First and second cell support scaffolds are positioned within the chamber, each scaffold comprising at least one three-dimensional porous matrix containing non woven fibrous polyethylene terephthalate, the first scaffold being positioned within the chamber between the upper channel and the middle channel, and the second scaffold being positioned within the chamber between the middle channel and the lower channel.

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Tallahassee, FL, United States of America

Teng Ma