Comparison of decellularization protocols for cultured cell-derived extracellular matrix-Effects on decellularization efficacy, extracellular matrix retention, and cell functions.

The in vitro reconstruction of the extracellular matrix (ECM) is required in tissue engineering and regenerative medicine because the ECM can regulate cell functions in vivo. For ECM reconstruction, a decellularization technique is used. ECM reconstructed by decellularization (dECM) is prepared from tissues/organs and cultured cells. Although decellularization methods have been optimized for tissue-/organ-derived dECM, the methods for cultured cell-derived dECM have not yet been optimized. Here, two physical (osmotic shocks) and five chemical decellularization methods are compared. The decellularization efficacies were changed according to the decellularization methods used. Among them, only the Triton X-100 and Tween 20 treatments could not decellularize completely. Additionally, when the efficacies were compared among different types of cells (monolayered cells with/without strong cell adhesion, multilayered cells), the efficacies were decreased for multilayered cells or cells with strong cell adhesion. Retained ECM contents tended to be greater in the dECM prepared by osmotic shocks than in those prepared by chemical methods. The contents impacted cell adhesion, shapes, growth and intracellular signal activation on the dECM. The comparison would be helpful for the optimization of decellularization methods for cultured cells, and it could also provide new insights into developing milder decellularization methods for tissues and organs.

A decellularized extracellular matrix derived from keratinocytes can suppress cellular senescence induced by replicative and oxidative stresses.

Cellular senescence is one of the barriers to maintain in vitro three-dimensional (3D) epidermal models for a long period of time. Therefore, a new culture substrate should be developed to suppress keratinocyte senescence to establish an epidermal model. In this study, reconstituted extracellular matrices (ECM) were prepared by culturing keratinocytes at different passages using the decellularization technique. The ECM prepared by decellularization (dECM) supports keratinocyte adhesion and growth. It has also been demonstrated that the dECM suppresses keratinocyte senescence by increasing the antioxidant activity. In particular, the dECM derived from younger passaged keratinocytes suppresses senescence more significantly than the dECM derived from highly passaged keratinocytes. Moreover, the dECM derived from younger passaged keratinocytes can suppress keratinocyte senescence during passage culture. Finally, the dECM derived from younger passaged keratinocytes increased AQP3 gene expression as an indicator of the functions of basal keratinocytes and the AQP3 expression ability to respond to all-trans retinoic acid. The dECM derived from younger passaged keratinocytes could be a useful culture substrate for developing an in vitro epidermal model.

Decellularized Extracellular Matrix for Cell Biology.

The extracellular matrix (ECM) is an architecture that supports the cells in our bodies and regulates various cell functions. The ECM is composed of many proteins and carbohydrates, and these molecules activate various intracellular signaling pathways orchestrated to decide cell fates. Therefore, it is not enough to study the role of single ECM molecules to understand the roles of the ECM in the regulation of cell functions; it is necessary to understand how the ECM, as an assembly of various molecules, regulates cell functions as a whole. For this purpose, in vitro ECM models mimicking native ECM are required. Here, a decellularization technique is presented to reconstitute native ECM in vitro. In this article, methods for preparing decellularized ECM (dECM) are described for use in tumor and stem cell biology. Additionally, a method for confirmation of decellularization and a dECM modification method are described. These dECM types will be useful for comprehensive studies of ECM roles. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of in vitro extracellular matrix (ECM) models mimicking native ECM in different malignant tumor tissues Basic Protocol 2: Preparation of in vitro ECM models mimicking native ECM surrounding myoblasts differentiating into myotubes at each myogenic stage Support Protocol 1: Confirmation of myogenic stages by myogenic stages by myogenic gene expression analysis Basic Protocol 3: Confirmation of cell removal Basic Protocol 4: Reduction of chondroitin sulfate chains in cultured cell-derived decellularized ECM Support Protocol 2: Quantification of chondroitin sulfate chain amounts in the decellularized ECM.

Decellularized extracellular matrices derived from cultured cells at stepwise myogenic stages for the regulation of myotube formation.

The regulation of stem cell differentiation is key for muscle tissue engineering and regenerative medicine. To this end, various substrates mimicking the native extracellular matrix (ECM) have been developed with consideration of the mechanical, topological, and biochemical properties. However, mimicking the biochemical properties of the native ECM is difficult due to its compositional complexity. To develop substrates that mimic the native ECM and its biochemical properties, decellularization is typically used. Here, substrates mimicking the native ECM at each myogenic stage are prepared as stepwise myogenesis-mimicking matrices via the in vitro myogenic culture of C2C12 myoblasts and decellularization. Cells adhered to the stepwise myogenesis-mimicking matrices at similar levels. However, the matrices derived from cells at the myogenic early stage suppressed cell growth and promoted myogenesis. This promotion of myogenesis was potentially due to the suppression of the activation of endogenous BMP signaling following the suppression of the expression of the myogenic-inhibitory factors, Id2 and Id3. Our stepwise myogenesis-mimicking matrices will be suitable ECM models for basic biological research and myogenesis of stem cells. Further, these matrices will provide insights that improve the efficacy of decellularized ECM for muscle repair.

Decellularized Extracellular Matrix for Cancer Research.

Genetic mutation and alterations of intracellular signaling have been focused on to understand the mechanisms of oncogenesis and cancer progression. Currently, it is pointed out to consider cancer as tissues. The extracellular microenvironment, including the extracellular matrix (ECM), is important for the regulation of cancer cell behavior. To comprehensively investigate ECM roles in the regulation of cancer cell behavior, decellularized ECM (dECM) is now used as an in vitro ECM model. In this review, I classify dECM with respect to its sources and summarize the preparation and characterization methods for dECM. Additionally, the examples of cancer research using the dECM were introduced. Finally, future perspectives of cancer studies with dECM are described in the conclusions.

An extracellular matrix (ECM) model at high malignant colorectal tumor increases chondroitin sulfate chains to promote epithelial-mesenchymal transition and chemoresistance acquisition.

Chemoresistance is one of the major barriers for tumor chemotherapy. It is clinically known that chemoresistance increases during tumor progression. Additionally, the extracellular matrix (ECM) is also remodeled during tumor progression. However, it remains unclear how ECM remodeling contributes to chemoresistance acquisition. Recently, it has been reported that epithelial-mesenchymal transition (EMT) contributes to chemoresistance acquisition. Here, how ECM remodeling contributes to 5-fluorouracil (5-FU) resistance acquisition was investigated from the viewpoints of EMT using in vitro ECM models mimicking native ECM in colorectal tumor tissue at three different malignant levels. 5-FU partially induced EMT and increased ABCB1 in colorectal HT-29 cells via TGF-ß signaling (an invasive tumor cell model). When HT-29 cells were cultured on an ECM model (high malignant matrices) mimicking native ECM in highly malignant tumor tissues, the cells facilitated TGF-ß-induced EMT and increased ABCB1 upregulation compared with that of other ECM models mimicking the low malignant level and normal tissues. High malignant matrices contained more chondroitin sulfate (CS) chains than those of other ECM models. Finally, CS chain-reduced high malignant matrices could not facilitate ABCB1 upregulation and TGF-ß-induced EMT. These results demonstrated that ECM remodeling during tumor progression increased CS chains to facilitate EMT and ABCB1 upregulation, contributing to chemoresistance acquisition.

Characterization of Initial Cell Adhesion on Charged Polymer Substrates in Serum-Containing and Serum-Free Media.

Charged substrates are expected to promote cell adhesion via electrostatic interaction, but it remains unclear how cells adhere to these substrates. Here, initial cell adhesion (<30 min) was re-examined on charged substrates in serum-containing and serum-free media to distinguish among various cell adhesion mechanisms (i.e., electrostatic interaction, hydrophobic interaction, and biological interaction). Cationic and anionic methacrylate copolymers were coated on nonionic nontissue culture-treated polystyrene to create charged substrates. Cells adhered similarly on cationic, anionic, and nonionic substrates in serum-free medium via integrin-independent mechanisms, but their adhesion forces differed (anionic > cationic > nonionic substrates), indicating that cell adhesion is not mediated solely by the cells' negative charge. In serum-containing medium, the cells adhered minimally on anionic and nonionic substrates, but they adhered abundantly on cationic substrates via both integrin-dependent and -independent mechanisms. These results suggest that neither electrostatic force nor protein adsorption is accountable for cell adhesion. Conclusively, the observed phenomena revealed a gap in the generally accepted understanding of cell adhesion mechanisms on charged polymeric substrates. A reanalysis of their mechanisms is necessary.

Preparation of Cell-Derived Decellularized Matrices Mimicking Native ECM During the Osteogenesis and Adipogenesis of Mesenchymal Stem Cells.

The extracellular matrix (ECM) is an important extracellular microenvironmental factor that regulates stem cell differentiation. The ECM is remodeled according to stem cell differentiation progression to precisely regulate the differentiation. Thus, it is expected that the matrices mimicking native ECM surrounding differentiating cells at each differentiation stage provide a favorable microenvironment to promote stem cell differentiation. However, it is difficult to prepare matrices mimicking native ECM using chemical methods because the ECM has a complicated composition. The decellularization technique is useful to prepare such matrices. In this chapter, we described the protocol to prepare matrices mimicking native ECM surrounding cells that are differentiating from mesenchymal stem cells to either osteoblasts or adipocytes via stem cell differentiation culture and a detergent- and nuclease-based decellularization technique.

Maintenance of Cartilaginous Gene Expression of Serially Subcultured Chondrocytes on Poly(2-Methoxyethyl Acrylate) Analogous Polymers.

Chondrocytes are important for cartilage tissue engineering. However, dedifferentiation during chondrocyte subculture prevents the application of cartilage tissue engineering. Therefore, prevention of this dedifferentiation is required. Here, the possibility of poly(2-methoxyethyl acrylate) (PMEA) and its analogous polymers, poly(tetrahydrofurfuryl acrylate) (PTHFA) and poly(2-(2-methoxyethoxy) ethyl acrylate-co-butyl acrylate) (PMe2A), for chondrocyte subculture without dedifferentiation is examined. Chondrocytes spread on PTHFA and polyethylene terephthalate (PET), whereas their spreading is delayed on PMEA and PMe2A. When primary chondrocytes are subcultured on these polymers, the expression levels of cartilaginous genes are higher on PMEA and PMe2A than on PET and PTHFA. Integrin contribution to the initial cell adhesion is lower on PMEA and PMe2A than on PTHFA and PET. This low level of integrin contribution to cell adhesion may cause a delay in cell spreading and the maintenance of cartilaginous gene expression. These results indicate that PMEA and PMe2A may be favorable substrates for chondrocyte subculture and cartilage tissue engineering.

Evaluation of initial cell adhesion on poly (2-methoxyethyl acrylate) (PMEA) analogous polymers.

Cell adhesion is a major concern in biomaterial development. Generally, cells adhere to polymeric substrates via the interaction between integrins and proteins adsorbed on the substrates. Previously, it was reported that poly (2-methoxyethyl acrylate) (PMEA) and its analogous polymers can alter the integrin dependency for cell adhesion. In particular, integrin-independent adhesion was observed on PMEA. However, initial adhesion mechanisms, including integrin-independent adhesion mechanisms, on PMEA are not well characterized. In this study, initial cell adhesion within 10 min was characterized on PMEA analogous polymers. Protein adsorption was suppressed on PMEA compared with tissue culture polystyrene, but the cell adhesion site in adsorbed fibronectin was exposed to the cells similarly. HT-1080 cells adhered on PMEA in a serum medium even in the presence of EDTA, suggesting that the cells adhered via both integrin-dependent and integrin-independent mechanisms. Finally, the cell adhesion force was measured by single-cell force spectroscopy. The cell adhesion force was not changed on PMEA in serum and serum-free media, suggesting that the cells adhered on PMEA directly. In conclusion, the control of protein adsorption is useful for regulating integrin dependency for cell adhesion and following the expression of cell functions regulated by integrins.

Integrin-independent Cell Adhesion Substrates: Possibility of Applications for Mechanobiology Research.

Cells can mainly sense mechanical cues from the extracellular matrix via integrins. Because mechanical cues can strongly influence cellular functions, understanding the roles of integrins in the sensing of mechanical cues is a key for the achievement of tissue engineering. The analyses to determine the roles of integrins in the sensing of mechanical cues have been performed by many methods based on molecular- and cell-biological techniques, atomic force microscopy, and optical tweezers. Integrin-dependent cell adhesion substrates have been also used for this purpose. Additionally, the cells can adhere on several substrates via integrin-independent mechanisms. There are two types of integrin-independent cell adhesion substrates; 1) the substrates immobilized with ligands against the receptors on cell surface and 2) the substrates suppressing protein adsorption. Cells can exhibit specific functions on these substrates. Here, the examples of integrin-independent cell adhesion substrates were reviewed, and their possible applications in mechanobiology research are discussed.

Promotion of Adipogenesis of 3T3-L1 Cells on Protein Adsorption-Suppressing Poly(2-methoxyethyl acrylate) Analogs.

Stem cell differentiation is an important issue in regenerative medicine and tissue engineering. It has been reported that cell shape is one of the factors that determine the lineage commitment of mesenchymal stem cells (MSCs). Therefore, the substrates have been developed to control their shapes. Recently, we found that poly(2-methoxyethyl acrylate) (PMEA) analogs can control tumor cell shape through the alteration of protein adsorption. Here, the adipogenesis of an adipocyte-progenitor cell, 3T3-L1 cells, was attempted; adipogenesis was to be regulated by surfaces coated with PMEA analogs through the control of their shape. The adipogenesis of 3T3-L1 cells was promoted on the surfaces coated with PMEA and its analogs, PMe3A and PMe2A. Evident focal adhesions were hardly observed on these surfaces, suggesting that integrin signal activation was suppressed. Additionally, actin assembly and cell spreading were suppressed on these surfaces. Therefore, the surfaces coated with PMEA analogs are expected to be suitable surfaces to regulate adipogenesis through the suppression of cell spreading. Additionally, we found that protein adsorption correlated with actin assembly and adipogenesis.

Decellularized matrices as in vitro models of extracellular matrix in tumor tissues at different malignant levels: Mechanism of 5-fluorouracil resistance in colorectal tumor cells.

Chemoresistance is a major barrier for tumor chemotherapy. It is well-known that chemoresistance increases with tumor progression. Chemoresistance is altered by both genetic mutations and the alteration of extracellular microenvironment. Particularly, the extracellular matrix (ECM) is remodeled during tumor progression. Therefore, ECM remodeling is expected to cause the acquisition of chemoresistance in highly malignant tumor tissue. Here, we prepared cultured cell-derived decellularized matrices that mimic native ECM in tumor tissues at different stages of malignancy, and 5-fluorouracil (5-FU) resistance was compared among these matrices. 5-FU resistance of colorectal tumor cells increased on the matrices derived from highly malignant tumor HT-29 cells, although the resistance did not increase on the matrices derived from low malignant tumor SW480 cells and normal CCD-841-CoN cells. The resistance on HT-29 cell-derived matrices increased through the activation of Akt and the upregulation of ABCB1 and ABCC1 without cell growth promotion, suggesting that ECM remodeling plays important roles in the acquisition of chemoresistance during tumor progression. It is expected that our decellularized matrices, or "staged tumorigenesis-mimicking matrices", will become preferred cell culture substrates for in vitro analysis of comprehensive ECM roles in chemoresistance and the screening and pharmacokinetic analysis of anti-cancer drugs.

Matrices secreted during simultaneous osteogenesis and adipogenesis of mesenchymal stem cells affect stem cells differentiation.

The extracellular matrix (ECM) plays a pivotal role in regulating stem cell functions. The ECM dynamically changes during tissue development. It remains a great challenge to mimic the dynamically changing ECM. In this study, we prepared novel types of extracellular matrices that could mimic the dynamic variation of extracellular matrices, which were derived from simultaneous osteogenesis and adipogenesis of human bone marrow-derived mesenchymal stem cells (MSCs). Four ECMs simultaneously mimicking early osteogenesis and early adipogenesis (EOEA), early osteogenesis and late adipogenesis (EOLA), late osteogenesis and early adipogenesis (LOEA), late osteogenesis and late adipogenesis (LOLA) were prepared. The stepwise osteogenesis-co-adipogenesis-mimicking matrices had different compositions and different effects on the osteogenic and adipogenic differentiation of MSCs. The matrices could provide very useful tools to investigate the interaction between ECM and stem cells and the role of ECM on stem cell differentiation. STATEMENT OF SIGNIFICANCE: Extracellular matrices (ECMs) are dynamically remodeled to regulate stem cell functions during tissue development. Until now, mimicking the ECM variation during stem cell differentiation to single cell type has been reported. However, there is no report on simultaneous mimicking of stem cell differentiation to two types of cells. In this study, we prepared the mixture ECMs derived from simultaneous osteogenesis and adipogenesis of MSCs at different stages and found that they could regulate stem cell differentiation. The concept is new and the ECMs are novel. No such ECMs have been reported previously. The matrices will provide very useful tools to investigate the interaction between ECM and stem cells and the role of ECM on stem cell differentiation.

Decellularized Extracellular Matrix as an In Vitro Model to Study the Comprehensive Roles of the ECM in Stem Cell Differentiation.

Stem cells are a promising cell source for regenerative medicine. Stem cell differentiation must be regulated for applications in regenerative medicine. Stem cells are surrounded by extracellular matrix (ECM) in vivo. The ECM is composed of many types of proteins and glycosaminoglycans that assemble into a complex structure. The assembly of ECM molecules influences stem cell differentiation through orchestrated intracellular signaling activated by many ECM molecules. Therefore, it is important to understand the comprehensive role of the ECM in stem cell differentiation as well as the functions of the individual ECM molecules. Decellularized ECM is a useful in vitro model for studying the comprehensive roles of ECM because it retains a native-like structure and composition. Decellularized ECM can be obtained from in vivo tissue ECM or ECM fabricated by cells cultured in vitro. It is important to select the correct decellularized ECM because each type has different properties. In this review, tissue-derived and cell-derived decellularized ECMs are compared as in vitro ECM models to examine the comprehensive roles of the ECM in stem cell differentiation. We also summarize recent studies using decellularized ECM to determine the comprehensive roles of the ECM in stem cell differentiation.

Regulation of the Contribution of Integrin to Cell Attachment on Poly(2-Methoxyethyl Acrylate) (PMEA) Analogous Polymers for Attachment-Based Cell Enrichment.

Cell enrichment is currently in high demand in medical engineering. We have reported that non-blood cells can attach to a blood-compatible poly(2-methoxyethyl acrylate) (PMEA) substrate through integrin-dependent and integrin-independent mechanisms because the PMEA substrate suppresses protein adsorption. Therefore, we assumed that PMEA analogous polymers can change the contribution of integrin to cell attachment through the regulation of protein adsorption. In the present study, we investigated protein adsorption, cell attachment profiles, and attachment mechanisms on PMEA analogous polymer substrates. Additionally, we demonstrated the possibility of attachment-based cell enrichment on PMEA analogous polymer substrates. HT-1080 and MDA-MB-231 cells started to attach to poly(butyl acrylate) (PBA) and poly(tetrahydrofurfuryl acrylate) (PTHFA), on which proteins could adsorb well, within 1 h. HepG2 cells started to attach after 1 h. HT-1080, MDA-MB-231, and HepG2 cells started to attach within 30 min to PMEA, poly(2-(2-methoxyethoxy) ethyl acrylate-co-butyl acrylate) (30:70 mol%, PMe2A) and poly(2-(2-methoxyethoxy) ethoxy ethyl acrylate-co-butyl acrylate) (30:70 mol%, PMe3A), which suppress protein adsorption. Moreover, the ratio of attached cells from a cell mixture can be changed on PMEA analogous polymers. These findings suggested that PMEA analogous polymers can be used for attachment-based cell enrichment.

Blood-Compatible Polymer for Hepatocyte Culture with High Hepatocyte-Specific Functions toward Bioartificial Liver Development.

The development of bioartificial liver (BAL) is expected because of the shortage of donor liver for transplantation. The substrates for BAL require the following criteria: (a) blood compatibility, (b) hepatocyte adhesiveness, and (c) the ability to maintain hepatocyte-specific functions. Here, we examined blood-compatible poly(2-methoxyethyl acrylate) (PMEA) and poly(tetrahydrofurfuryl acrylate) (PTHFA) (PTHFA) as the substrates for BAL. HepG2, a human hepatocyte model, could adhere on PMEA and PTHFA substrates. The spreading of HepG2 cells was suppressed on PMEA substrates because integrin contribution to cell adhesion on PMEA substrate was low and integrin signaling was not sufficiently activated. Hepatocyte-specific gene expression in HepG2 cells increased on PMEA substrate, whereas the expression decreased on PTHFA substrates due to the nuclear localization of Yes-associated protein (YAP). These results indicate that blood-compatible PMEA is suitable for BAL substrate. Also, PMEA is expected to be used to regulate cell functions for blood-contacting tissue engineering.

The Relationship Between Water Structure and Blood Compatibility in Poly(2-methoxyethyl Acrylate) (PMEA) Analogues.

Six types of poly(2-methoxyethyl acrylate) (PMEA) analogues were synthesized and the water structure in the hydrated polymers was characterized using differential scanning calorimetry (DSC). The hydrated PMEA analogues exhibited the different amounts of intermediate water. Non-thrombogenicity evaluation was performed on PMEA analogues for platelet adhesion and protein adsorption. Platelet adhesion was suppressed on PMEA analogues. In addition, the protein adsorption and deformation were suppressed by increasing the amount of intermediate water. This study demonstrates that the amount of intermediate water might play a key role in expressing the blood compatibility of polymeric materials.

Optimization of the tissue source, malignancy, and initial substrate of tumor cell-derived matrices to increase cancer cell chemoresistance against 5-fluorouracil.

The low chemoresistance of in vitro cancer cells inhibits the development of new anti-cancer drugs. Thus, development of a new in vitro culture system is required to increase the chemoresistance of in vitro cancer cells. Tumor cell-derived matrices have been reported to increase the chemoresistance of in vitro cancer cells. However, it remains unclear how tissue sources and the malignancy of cells used for the preparation of matrices affect the chemoresistance of tumor cell-derived matrices. Moreover, it remains unclear how the initial substrates used for the preparation of matrices affect the chemoresistance. In this study, we compared the effects of tissue sources and the malignancy of tumor cells, as well as the effect of the initial substrates on chemoresistance against 5-fluorouracil (5-FU). The chemoresistance of breast and colon cancer cells against 5-FU increased on matrices prepared with cells derived from the corresponding original tissues with higher malignancy. Moreover, the chemoresistance against 5-FU was altered on matrices prepared using different initial substrates that exhibited different characteristics of protein adsorption. Taken together, these results indicated that the appropriate selection of tissue sources, malignancy of tumor cells, and initial substrates used for matrix preparation is important for the preparation of tumor cell-derived matrices for chemoresistance assays.

Stem cell culture using cell-derived substrates.

There have been great efforts to develop cell culture systems to regulate stem cell functions. Development of cell culture substrates is one of the important approaches for stem cell culture because substrates influence stem cell functions such as attachment, proliferation, self-renewal, and induction of differentiation. Stem cells are surrounded by their specific microenvironments in vivo, composed of cells, cytokines, and an extracellular matrix (ECM), which may dynamically change and affect cellular activities accordingly. To mimic such microenvironments, cell culture substrates can be prepared by coating bioactive proteins such as ECM proteins and signaling molecules as ligands for cell surface receptors. Compared with protein-coated substrates, cell- and cell-formed ECM-derived substrates have shown great progress and attracted significant attention as functional and prospective biomaterials for stem cell culture and regenerative medicine. In this review, we summarize the latest progress of these new substrates derived from cells and cell-formed ECMs.