Dewaxed ECM: A simple method for analyzing cell behaviour on decellularized extracellular matrices

Abstract

Decellularization techniques have been used on a wide variety of tissues to create cell-seedable scaffolds for tissue engineering. Finding a suitable decellularization protocol for a certain type of tissue can be laborious, especially when organ perfusion devices are needed. In this study, we report a quick and simple method for comparing decellularization protocols combining the use of paraffin slices and two-dimensional cell cultures. We developed three decellularization protocols for adult murine kidney that yielded decellularized extracellular matrices (ECMs) with varying histological properties. The resulting paraffin-embedded ECM slices were deparaffinized and reseeded with murine embryonic stem cells (mESCs). We analyzed cell attachment four days post seeding via determination of cell numbers, and used quantitative Real-Time PCR 13 days post seeding to measure gene expression levels of two genes associated with renal development, Pax2 and Pou3f3. The three decellularization protocols produced kidney-matrices that showed clearly distinguishable results. We demonstrated that formerly paraffin-embedded decellularized ECMs can effectively influence differentiation of stem cells. This method can be used to identify optimal decellularization protocols for recellularization of three-dimensional tissue-scaffolds with embryonic stem cells and other tissue-specific cell types.

Strategies to develop endogenous stem cell-recruiting bioactive materials for tissue repair and regeneration

“…Similarly, the use of acid or alkaline treatments may cause a degradation in the major components of the ECM while ionic detergent may disrupt electrostatic interactions among proteins and be difficult to remove after the process is completed. All these aspects can affect the process of stem cell recruitment and differentiation toward a defined cell lineage [110]. A more detail discussion regarding the topic of decellularized ECM tissues for cardiac and bone tissue regeneration will be provided in the next sections.…”

Controlling stem cell behavior with decellularized extracellular matrix scaffolds

“…All of these factors can also affect how cells interact with the materials because they change the environment with which the cells are in direct contact. Therefore, care must be taken to choose the ideal decellularization protocol for each application because it can affect the success of directing cells towards a specific lineage [9]. In short, considerations must be made in designing and selecting the optimal decellularization process before the materials are manipulated into their final form.…”

“…The two main approaches for creating these slices are either to first decellularize the entire organ then cut it into slices or do the opposite by sectioning the unprocessed organ and then decellularizing the slices. To section the organ after decellularization, the organ must first be stiffened either by embedding the organ in agarose, OCT freezing medium, or paraffin [9–12, 23, 24]. However, the most common approach to create decellularized sections is to first cut the tissue into the desired thickness and then decellularize the slices of tissue.…”

Advances in the Knowledge about Kidney Decellularization and Repopulation

“…More specifically, it may employ detergents, salts, enzymes, and/or physical agents for the removal of tissue and organ cells. There are many existing methods for different applications (Gilbert et al, 2006; Badylak et al, 2011; Gilbert, 2012; Ofenbauer et al, 2015). In general, the process removes the cells present in the tissue, preserving partly the proteins of the ECM, such as collagen, with significantly reduced immunogenic potential (Mirmalek-Sani et al, 2013; Caralt et al, 2015).…”