Materials and methods for expansion of stem cells

a technology of stem cells and materials, applied in the field of stem cell expansion materials and methods, can solve the problems of reducing the therapeutic potency of hmsc transplantation, cell senescence, and low engraftment and homing efficiency of culture-expanded hmsc transplantation, so as to improve or enhance the therapeutic potency of cells, improve the translational success of cells, and enhance the therapeutic effect of stem cells

Inactive Publication Date: 2017-03-23
FLORIDA STATE UNIV RES FOUND INC
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  • Abstract
  • Description
  • Claims
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Benefits of technology

[0006]The subject invention concerns novel and translatable materials and methods for expansion of stem cells, such as MSC, that significantly improve the cells' translational success in the treatment of various conditions, such as ischemic cardiovascular and cerebral injuries. The subject invention utilizes cell self-aggregation in a microcarrier bioreactor as a non-genetic means to enhance stem cell therapeutic potency. The subject invention integrates a cell aggregation process in a scalable bioreactor system.
[0007]One aspect of the present invention concerns methods for expanding and growing stem cells, such as MSC, in a manner that provides for improved or enhanced therapeutic potency of the cells. In one embodiment of the method, thermally responsive microcarriers (TRMs) are utilized in conjunction with a scalable bioreactor system, such as the spinner flask bioreactor or a rocking bioreactor

Problems solved by technology

However, rapid hMSCs expansion has been found to induce cellular senescence and reduce their therapeutic potency.
Transplantation of culture-expanded

Method used

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  • Materials and methods for expansion of stem cells
  • Materials and methods for expansion of stem cells
  • Materials and methods for expansion of stem cells

Examples

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example 1

Actin-Mediated Contractility Influences the Assembly of Multicellular hMSC Aggregates

[0076]While the initial steps of aggregation require close contact and cell-cell adhesion via cadherin molecules, reorganization of actin cortical network is crucial in the establishment of a mature cell-cell contact (Amack and Manning 2012). To investigate the temporal effects of actin-mediated contractility in hMSC aggregate formation, hMSC cultures were treated with cytoD (1) in plastic culture for 2 days prior to cell detachment (e.g., 2D pretreatment) or (2) 12 hours after aggregate formation on ULA surfaces (i.e., 3D treatment). In 2D pretreatment, hMSC displayed dose-dependent response in which cytoD disrupted hMSC aggregation at concentration above 0.6 μM and prevented aggregate compaction at lower concentrations (FIGS. 5A and 5B). In 3D treatment, hMSC aggregates remained intact in cytoD concentration of 0.6 μM and exhibited dose-dependent reduction on compaction similar to those of 2D pret...

example 2

Actin Mediates Aggregate Compaction but not Viability and Caspase Expression

[0078]LPA is a naturally occurring bioactive phospholipid with multiple biological functions that include its ability to initiate cytoskeleton contraction by RhoA activation, promote cell survival and proliferation, and to enhance survival of hypoxia-challenged neonatal cardiomyocytes (Tigyi et al. 1994; Moolenaar 1995). LPA has also been identified as a novel survival factor and protects MSCs against hypoxia and serum deprivation-induced apoptosis (Chen et al. 2008a). Treatment of hMSC aggregates by LPA, however, has limited effects on aggregate compaction as well as viability at LPA concentration up to 10 μM (FIGS. 7A-7E).

[0079]The involvement of actin-myosin based contractility was investigated by treatment of aggregates with Y-27632, which inhibits the phosphorylation of Rho-associated kinase (ROCK) and prevents cell compaction (Chen et al. 2010). As expected, inhibition of ROCK kinase by 10 μM Y-27632 r...

example 3

Actin Mediates Aggregate Morphology, Interaction, and Spreading on Adherent Surfaces

[0082]Disruption of actin significantly alters hMSC morphology in the aggregates. As shown in FIGS. 10A-1, 10A-2, 10B and 10C, cells in the hMSC aggregates were tightly packed and spread with limited interstitial space at the boundary of the aggregate. In contrast, cells in the cytoD- and Y-27632-treated aggregates were loosely packed and exhibited spherical morphology with abundant interstitial space as shown by SEM and histology (FIGS. 10A-1 and 10A-2). Histological sectioning also revealed contrasting morphology of hMSC in the interior of control and cytoD and Y-27632-treated aggregates. In the control aggregates, hMSCs are morphologically heterogeneous with spindle-shaped cells at the outer boundary and round and tightly packed cells in the interior, indicating morphological polarization. In the cytoD and Y-27632 treated aggregates, cells are loosely packed with no spreading at the boundary, indi...

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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.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]The present application claims the benefit of U.S. Provisional Application Ser. No. 61 / 946,415, filed Feb. 28, 2014, which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, or drawings.BACKGROUND OF THE INVENTION[0002]Human mesenchymal stem or stromal cells (hMSCs) have been tested in over 400 clinical trials in a wide range of diseases (clinicaltrials.gov). As hMSCs translate into clinics, there is an increasing demand on technology for scalable production of therapeutically competent hMSCs in scalable bioreactor system. However, rapid hMSCs expansion has been found to induce cellular senescence and reduce their therapeutic potency. As a result, hMSC expansion technology must not only meet the demand in quantity but also preserve hMSC therapeutic potency during expansion.[0003]Recent studies have shown that hMSC have unique properties to self-assemble i...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A61K35/28C12M3/04C12N5/00C12M3/06
CPCC12N5/0663C12N2500/42C12N5/0075C12M27/16C12M27/10A61K35/28C12N2500/02C12N2509/00C12N2513/00C12N2527/00C12N2533/30C12N2533/54C12N2533/70C12N2531/00C12N2501/33C12N2501/39C12N2501/998C12N5/0062C12N2501/727C12N2501/999C12N2539/10C12M25/16C12M33/00
Inventor MA, TENG
Owner FLORIDA STATE UNIV RES FOUND INC
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