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

Abstract

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.

Immunomodulatory functions of human mesenchymal stromal cells are enhanced when cultured on HEP/COL multilayers supplemented with interferon-gamma

“…(HEP/COL) multilayers were constructed as described in our previous works [ 12 , 29 , 39 , 40 ]. Heparin sodium (HEP) purchased from Celsus Laboratories, Inc. (Cat.…”

“…A 50 ​ng/mL concentration for soluble IFN-γ was selected based on our previous study [ 25 , 26 ]. Time points and the initial number of cells were selected according to the nature of the specific method used [ 40 ].…”

“…QCM-D measurements were performed on a quartz crystal microbalance with dissipation from Biolin Scientific, Sweden. The multilayer build-up process was described in our previous work [ 40 ]. Briefly, the quartz crystal was immersed in 5:1:1 (volume parts) at 75 ​°C of water, 25% ammonia, and 30% hydrogen peroxide.…”

“…#A13261) was used. hMSCs (10000 ​cells/cm 2 ) were seeded on each surface prepared on a 96 well-plate, and cell viability was measured after 3 days of culture as described in our previous works [ 29 , 40 ]. Briefly, the cell culture medium was removed after 3 days, and 100 ​μL per well containing 90% fresh cell medium and 10% PrestoBlue reagent were added.…”

“…#300600840, cell growth area of 0.64 ​cm 2 per well), which are composed of tissue culture plastic, but they also contain sensors to measure impedance, and hMSCs at a concentration of 20000 ​cells/cm 2 were seeded on each condition evaluated; uncoated sensors, multilayers ending in HEP, and ending in COL with and without IFN-γ supplemented in the culture medium. xCELLigence instrument was configured as described in our previous works [ [40] , [41] ]. Briefly, the xCELLigence RTCA S16 was placed inside the incubator to allow the S16 device to warm up for at least 2 ​h before use.…”