Selection of the optimal protocol for preparation of decellularized extracellular matrix of human adipose tissue-derived mesenchymal stromal cells

Currently, biological scaffolds composed of extracellular matrix (ECM) are being examined actively for the needs of regenerative medicine. ECM substrates are prepared by decellularization and used to deliver cells to damaged tissue. Native scaffold 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 multipotent potential of differentiation and secrete a wide range of bioactive molecules. In this regard, MSCs are important intermediaries for tissue repair. The ECM as a critical component of the MSC niche modulating their functional activity, including migration, proliferation and differentiation, as well as 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/NHOH solution in phosphate buffered solution or HO, and the possibility of using dcECM after spheroids were formed. ECM-derived substrates were analyzed with immunocytochemistry and scanning electron microscopy. During long-term сultivation MSСs prodded a well-developed EСM, which after treatment with phosphate buffered solution of Triton X-100/NH4OH maintaining the struсture dose to native one. 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. We detected fragments of ECM and cells after spheroids formation upon of RGD peptides treatment. Therefore, this method was not effective for obtaining dcECM of adMSCs.

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