Development of a Mesenchymal Stem-Cell Expansion Device – A Stem-Cell Bioreactor, 10-R9864

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Principal Investigator
Jian Ling

Inclusive Dates:  10/01/08 – 04/01/10

Background - The mesenchymal stem cells (MSCs) present in adult bone marrow are multipotent stem cells that can be used in the repair or regeneration of various tissues (including bone, cartilage, tendon, etc.). These tissues may become damaged or lost as a result of a variety of degenerative, age-related and traumatic causes. However, the sources of MSCs for tissue repair are limited because of their low population (0.001 percent) in adult bone marrow. Therefore, in vitro expansion of MSCs is needed for MSC-based therapy. The major challenge, however, is that the MSCs lose their multipotency (or their "stemness") as they are expanded in vitro using the conventional cell culture system. The loss of stemness will prevent these cells from differentiating into the needed cell types for tissue repair and therefore significantly reduce the efficacy of the MSC-based therapies. The objective of this project is to develop an in vitro MSC expansion device (also called a bioreactor) that mimics the bone marrow environment to promote MSC expansion while preserving multipotent properties.

Approach - To mimic the in vivo bone marrow environment, SwRI researchers developed 1) a three-dimensional (3D) collagen scaffold that has an open-cell interconnected porous network that resembles the trabecular bone structure, 2) a layer of bone marrow stromal tissue extracellular matrix (ECM) on the 3D collagen scaffold to achieve the bone-marrow tissue specification, 3) a specialized circulation system, or a bioreactor, to provide the stem cells grown in the 3D scaffolds controlled fluid flow, nutrition supply, gas (O2, CO2) concentration, and temperature conditions similar to the bone marrow environment, and 4) the methods to evaluate the performance of the bioreactor system in promoting MSC expansion and retaining MSC multipotent properties.

Accomplishments - Three-dimensional collagen scaffolds (Figure 1(a)) were created that have an open-cell interconnected porous structure. The average pore size was around 300 μm. Methods of physical, chemical cross-linking, and in vitro mineralization were used to enhance the mechanical strength of the scaffolds. A perfusion bioreactor system that controls flow, O2, CO2 concentrations, and supplies media through a 3D scaffold was established (Figure 1(b)). In addition, the bioreactor system allows the monitoring of cell activities on the 3D collagen scaffolds. Methods of seeding MSCs on collagen scaffolds and quantifying cell growth in the scaffolds were developed. MSCs were successfully cultured and proliferated in the collagen scaffold with the perfusion by the bioreactor system. Figure 1(c) illustrates live cells grown on the 3D collagen scaffolds. Time-lapse images were also used to monitor cell activities in a collagen scaffold (data not shown).

(a) Environmental scanning electron microscopy (ESEM) illustrates a collagen scaffold with the average pore size of 300 μm. (b) The perfusion flow bioreactor system includes the controls of flow rate, temperature, O2 and CO2 and a culture vessel that is suitable for live-cell imaging during cell cultivation. (c) A fluorescent image illustrates the live cells on a collagen scaffold (using Live/Dead assay).


Figure 1. (a) Environmental scanning electron microscopy (ESEM) illustrates a collagen scaffold with the average pore size of 300 μm. (b) The perfusion flow bioreactor system includes the controls of flow rate, temperature, O2 and CO2 and a culture vessel that is suitable for live-cell imaging during cell cultivation. (c) A fluorescent image illustrates the live cells on a collagen scaffold (using Live/Dead assay).


 

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