Selection of the Optimal Protocol for Preparation of a Decellularized Extracellular Matrix of Human Adipose Tissue-Derived Mesenchymal Stromal Cells

Аннотация

Currently, biological scaffolds composed of extracellular matrix (ECM) are being actively examined for the needs of regenerative medicine. ECM substrates are prepared by decellularization and used to deliver cells to damaged tissue. Native scaffolds of ECM have an advantage over bioengineered ones because ECM retains natural biologic cues that provide efficient reparative cell functions. Mesenchymal stromal cells (MSCs) have a multipotent potential of differentiation and secrete a wide range of bioactive molecules. In this regard, MSCs are valuable intermediaries for tissue repair. The ECM as a critical component of the MSCs niche modulates their functional activity, including migration, proliferation, and differentiation, and supports their potential for self-renewal. In vitro investigations would be useful in elucidation of how biological scaffolds can affect the reparative functions of MSCs. There are several different protocols for decellularization. Since ECM of various cell types differs qualitatively and quantitatively, these protocols should be optimized for each specific case. In the present study we compared the effectiveness of approach to prepare decellularized ECM (dcECM) of adipose-derived MSC (adMSC): Triton X-100/NH₄OH solution in phosphate buffered solution or H₂O, and the possibility of using dcECM after spheroids were formed. ECM-derived substrates were analyzed with immunocytochemistry and scanning electron microscopy. During long-term culture, MSСs produced a well-developed EСM, which maintained a structure close to the native one after treatment with phosphate buffered solution of Triton X‑100/NH₄OH. It was impossible to receive a uniform dcECM layer, when water solution of Triton X-100/NH₄OH was used. On the scanning electron microscopy images single fiber of ECM were revealed in this case. Fragments of ECM and cells after spheroids formation with RGD peptides were detected. Therefore, this method was not effective for obtaining dcECM of adMSCs.