Bioprocess development for cord blood mesenchymal stromal cells on microcarriers in Vertical-Wheel bioreactors.

Equine mesenchymal stromal cells (MSCs) have been found to be beneficial for the treatment of many ailments, including orthopedic injuries, due to their superior differentiation potential and immunomodulating properties. Cell therapies require large cell numbers, which are not efficiently generated using conventional static expansion methods. Expansion of equine cord blood-derived MSCs (eCB-MSCs) in bioreactors, using microcarriers as an attachment surface, has the potential to generate large numbers of cells with increased reproducibility and homogeneity compared with static T-flask expansion. This study investigated the development of an expansion process using Vertical-Wheel (VW) bioreactors, a single-use bioreactor technology that incorporates a wheel instead of an impeller. Initially, microcarriers were screened at small scale to assess eCB-MSC attachment and growth and then in bioreactors to assess cell expansion and harvesting. The effect of different donors, serial passaging, and batch versus fed batch were all examined in 0.1 L VW bioreactors. The use of VW bioreactors with an appropriate microcarrier was shown to be able to produce cell densities of up to 1E6 cells/mL, while maintaining cell phenotype and functionality, thus demonstrating great potential for the use of these bioreactors to produce large cell numbers for cell therapies.

Gene Expression Profile Is Different between Intact and Enzymatically Digested Equine Articular Cartilage.

OBJECTIVES: RNA isolation is necessary for the evaluation of gene expression. Due to the nature of its extracellular matrix, RNA isolation from articular hyaline cartilage is difficult and thus the tissue is commonly enzymatically digested in order to extract RNA from the obtained chondrocytes. We hypothesized that the digestion process affects the expression levels of common cartilage-associated genes. DESIGN: Expression of cartilage-associated genes was compared between intact cartilage and digested chondrocytes from weight bearing and non-weight bearing regions of the equine fetlock joint. RESULTS: The gene expression of SOX9, COL1A2, COL2A1, ACAN, and COLX were analyzed. Digested cartilage showed a significant decrease in the expression of COL1A2, COL2A1, and ACAN compared to intact cartilage in both joint regions, and an increase in COLX expression in non-weight bearing cartilage only. CONCLUSIONS: Enzymatic digestion of cartilage significantly impacts gene expression profile. We conclude that while RNA isolation from intact cartilage is more technically difficult, determination of gene expression should be conducted on intact cartilage if true representation of the in vivo processes is sought.

Equine Cord Blood Mesenchymal Stromal Cells Have Greater Differentiation and Similar Immunosuppressive Potential to Cord Tissue Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are the most common cell population studied for therapeutic use in veterinary medicine. MSCs obtained from neonatal sources such as umbilical cord tissue (CT-MSCs) or cord blood (CB-MSCs) are appealing due to the non-invasive nature of procurement and the time allowed for characterization of cells before use. However, it remains unclear as to whether CB- or CT-MSCs have equivalent progenitor and non-progenitor functions. CB-MSCs have been shown to have superior chondrogenic potential to MSCs from other sources, whereas their immunomodulatory capacity does not seem to vary significantly. Using equine CB-MSCs and CT-MSCs from the same donors, we hypothesized that MSCs from both sources would have a similar immunophenotype, that CB-MSCs would be more amenable to differentiation, and that they can equally suppress lymphocyte proliferation. We evaluated cells from both sources for "classic" equine MSC markers CD90, CD105, CD29, and CD44, as well as pericyte markers CD146, NG2, and α-SMA. Contrary to our hypothesis, CB-MSCs showed mid- to high expression of pericyte surface markers CD146 and NG2, whereas expression in CT-MSCs was absent. On trilineage differentiation, CB-MSCs were more osteogenic and chondrogenic based on alkaline phosphatase activity and glycosaminoglycan content, respectively. Finally, using a mononuclear cell (MNC) suppression assay, we determined that both CB-MSCs and CT-MSCs are capable of suppressing stimulated MNC proliferation to a similar degree. We have determined that the choice of MSC tissue source should be made with the intended application in mind. This appears to be particularly relevant if pursuing a progenitor-based treatment strategy.

Improved expansion of equine cord blood derived mesenchymal stromal cells by using microcarriers in stirred suspension bioreactors.

Equine mesenchymal stromal cells (MSCs) are increasingly investigated for their clinical therapeutic utility. Such cell-based treatments can require cell numbers in the millions or billions, with conventional expansion methods using static T-flasks typically inefficient in achieving these cell numbers. Equine cord blood-derived MSCs (eCB-MSCs), are promising cell candidates owing to their capacity for chondrogenic differentiation and immunomodulation. Expansion of eCB-MSCs in stirred suspension bioreactors with microcarriers as an attachment surface has the potential to generate clinically relevant numbers of cells while decreasing cost, time and labour requirements and increasing reproducibility and yield when compared to static expansion. As eCB-MSCs have not yet been expanded in stirred suspension bioreactors, a robust protocol was required to expand these cells using this method. This study outlines the development of an expansion bioprocess, detailing the inoculation phase, expansion phase, and harvesting phase, followed by phenotypic and trilineage differentiation characterization of two eCB-MSC donors. The process achieved maximum cell densities up to 75,000 cells/cm2 corresponding to 40 million cells in a 100 mL bioreactor, with a harvesting efficiency of up to 80%, corresponding to a yield of 32 million cells from a 100 mL bioreactor. When compared to cells grown in static T-flasks, bioreactor-expanded eCB-MSC cultures did not change in surface marker expression or trilineage differentiation capacity. This indicates that the bioreactor expansion process yields large quantities of eCB-MSCs with similar characteristics to conventionally grown eCB-MSCs.

Beyond Cartilage Repair: The Role of the Osteochondral Unit in Joint Health and Disease.

IMPACT STATEMENT: In this comprehensive review, we are providing a holistic overview of osteochondral tissue development, disease, pain localization, as well as structural evaluation and current repair strategies. This review is intended to serve as a broad introduction to this multidisciplinary research area. It is a thorough examination of the biological aspects of the osteochondral unit from a tissue engineering perspective, highlighting the importance of the subchondral bone in chondral and osteochondral lesion repair and pain relief.