A comparative evaluation of layer-by-layer assembly techniques for surface modification of microcarriers used in human mesenchymal stromal cell manufacturing.

The demand for large quantities of highly potent human mesenchymal stromal cells (hMSCs) is growing given their therapeutic potential. To meet high production needs, suspension-based cell cultures using microcarriers are commonly used. Microcarriers are commonly made of or coated with extracellular matrix proteins or charged compounds to promote cell adhesion and proliferation. In this work, a simple method (draining filter) to perform layer by layer (LbL) assembly on microcarriers to create multilayers of heparin and collagen and further demonstrate that these multilayers have a positive effect on hMSC viability after 48 h of culture was demonstrated. The draining filter method is evaluated against two other methods found in literature-centrifugation and fluidized bed, showing that the draining filter method can perform the surface modification with greater efficiency and with less materials and steps needed in the coating process.

Methods for the Assembly and Characterization of Polyelectrolyte Multilayers as Microenvironments to Modulate Human Mesenchymal Stromal Cell Response.

Thin films are of interest in materials design because they allow for the modification of surface properties of materials while the bulk properties of the material are largely unaffected. In this work, we outline methods for the assembly of thin films using a technique known as layer-by-layer (LbL). Furthermore, their interactions with human mesenchymal stromal cells (hMSCs) are discussed. hMSCs are a subject of growing interest because of their potential to treat or cure diseases, given their immunosuppressive properties, multipotent differentiation capabilities, and tissue regeneration capabilities. Numerous improvements and modifications have been suggested for the harvesting, treatment, and culture of hMSCs prior to their administration in human subjects. Here, we discuss methods to assess the interactions of hMSCs with thin LbL-assembled films of heparin and collagen. Three different methods are discussed. The first details the preparation of heparin/collagen multilayers on different surfaces and the seeding of cells on these multilayers. The second method details the characterization of multilayers, including techniques to assess the thickness, roughness, and surface charge of the multilayers, as well as in situ deposition of multilayers. The third method details the analysis of cell interactions with the multilayers, including techniques to assess proliferation, viability, real-time monitoring of hMSC behavior, analysis of hMSC-adhesive proteins on the multilayers, immunomodulatory factor expression of hMSCs, and cytokine expression on heparin/collagen multilayers. We propose that the methods described in this work will assist in the design and characterization of LbL-assembled thin films and the analysis of hMSCs cultured on these thin films.