Natural Extracellular Matrix Scaffold Research Articles

Comparative study of decellularization techniques to obtain natural extracellular matrix scaffolds of human peripheral-nerve allografts.

We hypothesize that adding sonication cycles to the process of decellularization of cadaveric human peripheral nerves will increase the removal of cell debris and myelin sheath, increasing their utility as allografts. Our aim of this study was to develop a decellularization protocol that allows the removal of cells and myelin sheath without detrimental effects on nerve architecture. Segments of ulnar and median nerves from human donors, isolated both before and after cardiac arrest, were subjected to two methods of decellularization: two-detergent-based (M1) and the same method with sonication added (M2). We evaluated the histology of unprocessed and decellularized nerves (n = 24 per group) for general morphology, presence of cell nuclei, nuclear remnants, collagen fibers, and myelin. We performed immunohistochemistry to verify the removal of Schwann cells associated with histomorphometry. We used scanning electron microscopy (EM) to evaluate the ultrastructure of both native and decellularized nerves. The efficacy of decellularization was assessed by analysis of genomic DNA. Histology confirmed that both decellularization protocols were adequate and maintained natural nerve architecture. Scanning EM showed that 3D ultrastructural architecture also was maintained. Histomorphometric parameters showed a more complete removal of the myelin with the M2 protocol than with M1 (p = 0.009). Fiber diameter and density were not modified by decellularization methods. Sonication can be a complementary method to decellularization of peripheral nerve allografts with sonication increasing the effectiveness of detergent-based protocols for the removal of unwanted cellular components from peripheral nerve allografts.

Protocols for the preparation and characterization of decellularized tissue and organ scaffolds for tissue engineering.

Extracellular matrix (ECM) scaffolds are extensively used in tissue engineering studies and numerous clinical applications for tissue and organ reconstructions. Due to the global severe shortage of human tissues and organs, xenogeneic biomaterials are a common sourcefor human tissue engineering and regenerative medicine applications. Traditional methods for decellularization often disrupt the 3D architecture and damage the structural integrity of the ECM scaffold. To efficiently obtain natural ECM scaffolds from animal tissues and organs with intact architecture, we have developed a platform decellularization process using supercritical CO2 and tested itspotential application in tissue engineering.A combination of human mesenchymal stem cells with adecellularized dermal matrix scaffold allowed complete regeneration of skin structure in a porcine full-thickness wound model.

Abstract 6315: High content screening of intestinal organoid cultures to visualize and quantify effects of drug combinations

Abstract Background: The choice in advanced pre-clinical in vitro models that use cultures of patient-derived tissues has increased but their full potential in drug discovery has not been fully exploited. Assays often lack sensitivity or clinically relevant end-points or are not suitable for high throughput screening. Here we describe a 3D screening platform that combines complex biology with high content phenotype-based image analysis to provide visualization and quantification of the effects of various targeted therapies on healthy and diseased tissue. Improved characterization of compound effects increases the scope for predicting treatment responses in patients, discriminating different drug responses and flagging off-target effects. Methods: A panel of tumor and healthy intestinal organoids, obtained from HUB Organoid Technology, were cultured in natural extracellular matrix scaffolds that support appropriate gene expression, differentiation and functional characteristics of the tissue of origin. To investigate the effects of anti-EGFR targeted therapies in combination with standard-of-care (SoC) treatments on the function, formation and integrity of colorectal cancer organoids (CRCs) and matching normal gut epithelium, we set up the following intestinal organoid test systems: 1) A panel of CRCs with a broad heterogeneity of mutations for high throughput phenotype-based screening that enable accurate compound profiling 2) Normal vs. tumor organoid pair assays for off-target effect studies and 3) Mode of action studies in various tumoroid models. Results: High content 3D image analysis of the organoids enabled sensitive detection of treatment-induced and compound-specific morphological changes beyond conventional cell viability measurements, including those associated with tumor killing, cell cycle arrest, toxicity and epithelial integrity to discriminate therapeutic and adverse responses in intestinal organoids. This enabled distinction between different mechanisms of action and determination of compound synergy. The image-based measurements can be complemented with detection of secreted factors (e.g. cytokines, chemokines) or expressed genes in response to various therapeutic compounds. Conclusion: The advanced 3D image analysis of in vitro cultured organoids described here represents a rapid and biologically relevant approach to test various anti-cancer-therapeutic modalities, including antibodies, antibody-drug conjugates and small molecules. This platform technology is suitable not only for high throughput screening in organoid panels but also in-depth mode of action studies. Moreover, evaluating effects on healthy and tumor intestinal organoids from the same patient and between patients, gives insight into potential toxicities, therapeutic window and patient-derived differences in subsequent in vivo studies. Citation Format: Mariusz Madej, Cinthya Del Angel Zuvirie, Jara García Mateos, Leo Price, Kuan Yan, Bram Herpers. High content screening of intestinal organoid cultures to visualize and quantify effects of drug combinations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6315.

Abstract A065: Differential sensitivity of normal and tumor organoids to targeted therapies

Abstract Background: Advances in the culture of cells from human tissues have enabled the generation of normal and tumor organoid pairs derived from the same patient. These organoid cultures increase the scope for predicting responses to novel cancer therapeutics in patients. Here we present a high throughput 3D human intestinal organoid culture platform combined with high content image-based analysis. This platform allows visualization and quantification of responses beyond conventional cell viability measurements, including those associated with tumor killing, cell cycle arrest, toxicity and epithelial permeability and discriminates therapeutic and adverse responses in healthy tissue and tumor. Methods: Intestinal organoids, obtained from HUB Organoid Technology, cultured in natural extracellular matrix scaffolds show gene expression patterns, differentiation and functional characteristics that closely resemble the in vivo biology. To investigate the effect of standard-of-care (SoC) treatments, novel targeting antibodies and small molecules on tumor growth suppression and killing as well as function, formation and integrity of the gut epithelium, we tested a panel of normal and colorectal cancer organoids with a broad heterogeneity of mutations. 3D imaging and analysis was performed and responses in normal vs. tumor were compared. Results: High content 3D image analysis of the organoid enabled sensitive detection of treatment-induced and compound-specific morphological changes such as (inhibition of) growth, development, lumen formation, epithelial integrity and cell death. This enabled distinction between mechanisms of action and distinct effects and sensitivities in normal epithelium and tumor tissue from the same patient. Conclusion: 3D image analysis of in vitro cultured organoids represents a rapid and biologically relevant approach to test various anti-cancer-therapeutic modalities, including antibodies, antibody-drug conjugates and small molecules. Evaluating effects on healthy and tumor tissue from the same patient can give insight into potential toxicities and therapeutic window. Citation Format: Mariusz Madej, Cinthya Del Angel Zuvirie, Jara García Mateos, Leo Price, Lidia Daszkiewicz, Kuan Yan, Bram Herpers. Differential sensitivity of normal and tumor organoids to targeted therapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A065. doi:10.1158/1535-7163.TARG-19-A065

Abstract 2083: Human normal and tumor colon organoids: a 3D high content screening platform for anticancer drug development

Abstract Background: Recent advances in establishing organoids from human specimens have enabled further development of advanced 3D cell culture models such as normal and tumor colon organoids derived from the same patient. These organoid cultures increase the scope for predicting treatment responses in patients, enabling the testing of cancer (immune)therapies, discriminating different drug responses and flagging off-target effects. Here we present a high throughput 3D human intestinal organoid culture platform combined with high content image-based analysis. This platform allows visualization and quantification of various treatment effects on healthy and diseased gut epithelium. Methods: Intestinal organoids, obtained from HUB Organoid Technology, are cultured in natural extracellular matrix scaffolds and show gene expression patterns, differentiation and functional characteristics that closely resemble the in vivo biology. To investigate the effect of standard-of-care (SoC) treatments like 5-FU, Oxaliplatin and Irinotecan and/or (in combination with) novel targeting antibodies or other small molecules on the function, formation and integrity of the gut epithelium, we set up the following intestinal organoid test systems: 1. Colorectal cancer organoids with a broad heterogeneity of mutations for sensitive and high throughput phenotypic screening that enable accurate compound profiling and 2. Human normal vs. tumor organoid pair assays for off-target effect studies. Results: High content 3D image analysis of the organoid models enables sensitive detection of treatment-induced and compound-specific morphological changes such as (inhibition of) growth, development, lumen formation, epithelial integrity and cell death. This enables distinction between mechanisms of action and determining compound synergy. The image-based measurements can be complemented with detection of secreted factors (e.g. cytokines, chemokines) or expressed genes in response to various therapeutic compounds. Conclusion: OcellO’s advanced 3D image analysis of in vitro cultured organoids represents a rapid and biologically relevant approach to test various candidate compounds (e.g. antibodies, antibody-drug conjugates and small molecules) that can treat intestinal disorders such as Inflammatory Bowel Disease and colon cancer. This organoid technology also allows identifying potential adverse effects that can occur during in-vivo studies. Therefore, our intestinal organoid screening platform represents a significant advance on conventional in vitro models and helps bridge the translational gap between in vivo studies. Note: This abstract was not presented at the meeting. Citation Format: Mariusz Madej, Jara Garcia Mateos, Kuan Yan, Lidia Daszkiewicz, Bram Herpers, Leo S. Price. Human normal and tumor colon organoids: a 3D high content screening platform for anticancer drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2083.

Acellular Mouse Kidney ECM can be Used as a Three-Dimensional Substrate to Test the Differentiation Potential of Embryonic Stem Cell Derived Renal Progenitors

The development of strategies for tissue regeneration and bio-artificial organ development is based on our understanding of embryogenesis. Differentiation protocols attempt to recapitulate the signaling modalities of gastrulation and organogenesis, coupled with cell selection regimens to isolate the cells of choice. This strategy is impeded by the lack of optimal in vitro culture systems since traditional culture systems do not allow for the three-dimensional interaction between cells and the extracellular matrix. While artificial three-dimensional scaffolds are available, using the natural extracellular matrix scaffold is advantageous because it has a distinct architecture that is difficult to replicate. The adult extracellular matrix is predicted to mediate signaling related to tissue repair not embryogenesis but existing similarities between the two argues that the extracellular matrix will influence the differentiation of stem and progenitor cells. Previous studies using undifferentiated embryonic stem cells grown directly on acellular kidney ECM demonstrated that the acellular kidney supported cell growth but limited differentiation occurred. Using mouse kidney extracellular matrix and mouse embryonic stem cells we report that the extracellular matrix can support the development of kidney structures if the stem cells are first differentiated to kidney progenitor cells before being applied to the acellular organ.

Natural extracellular matrix scaffolds recycled from human salivary digests: a morphometric study.

A challenge in engineering tissues is to supply parenchymal cells with suitable scaffolds which ideally reproduce the extracellular matrix (ECM). This study tested the hypothesis of preserving the 'residual connective tissue' remaining after mechanical and enzymatic release of cells from human submandibular gland biopsies (that we named 'natural ExtraCellular Matrix scaffolds', nECMsc) to be used as recycled natural scaffolds. The objective was to test whether nECMsc and native salivary tissue were comparable morphologically, in ECM proteins composition, and in cell seeding efficiency. Following cell isolation procedures, nECMsc were kept, either fresh or frozen (sectioned into 12-μm-thick slices), and examined with high-resolution electron microscopy (HRSEM) for its three-dimensional structure, and with picrosirius red staining and immunogold staining for ECM protein composition and distribution, respectively. nECMsc were seeded with human epithelial cells and fibroblasts to assess cell attachment and proliferation in short-term experiments. Under HRSEM, nECMsc had comparable fiber arrangement to original glands. Histochemical and immunogold-labeling examinations revealed the presence of collagen types I, III, and IV. Seeded epithelial cells and fibroblasts attached, proliferated (14-55%), and were alive (86-99%) after 4-8days of culture. nECMsc retained native ECM proteins and maintained their distribution. Seeded cells remained viable on nECMsc.

Decellularization of fibroblast cell sheets for natural extracellular matrix scaffold preparation.

The application of cell-derived extracellular matrix (ECM) in tissue engineering has gained increasing interest because it can provide a naturally occurring, complex set of physiologically functional signals for cell growth. The ECM scaffolds produced from decellularized fibroblast cell sheets contain high amounts of ECM substances, such as collagen, elastin, and glycosaminoglycans. They can serve as cell adhesion sites and mechanically strong supports for tissue-engineered constructs. An efficient method that can largely remove cellular materials while maintaining minimal disruption of ECM ultrastructure and content during the decellularization process is critical. In this study, three decellularization methods were investigated: high concentration (0.5 wt%) of sodium dodecyl sulfate (SDS), low concentration (0.05 wt%) of SDS, and freeze-thaw cycling method. They were compared by characterization of ECM preservation, mechanical properties, in vitro immune response, and cell repopulation ability of the resulted ECM scaffolds. The results demonstrated that the high SDS treatment could efficiently remove around 90% of DNA from the cell sheet, but significantly compromised their ECM content and mechanical strength. The elastic and viscous modulus of the ECM decreased around 80% and 62%, respectively, after the high SDS treatment. The freeze-thaw cycling method maintained the ECM structure as well as the mechanical strength, but also preserved a large amount of cellular components in the ECM scaffold. Around 88% of DNA was left in the ECM after the freeze-thaw treatment. In vitro inflammatory tests suggested that the amount of DNA fragments in ECM scaffolds does not cause a significantly different immune response. All three ECM scaffolds showed comparable ability to support in vitro cell repopulation. The ECM scaffolds possess great potential to be selectively used in different tissue engineering applications according to the practical requirement.

Effects of Natural Cartilaginous Extracellular Matrix on Chondrogenic Potential for Cartilage Cell Transplantation

Autologous chondrocyte transplantation (ACT) has been established to contribute cartilage regeneration over the past years; however, many obstacles need to be overcome. Recently, newer ACT technique involves cotransplantation of chondrocytes and biomaterial. Although various proposed intelligent biomaterials exist, many of them remain insufficient and controversial. In this study, we aimed to examine the effects of natural extracellular matrix (ECM) to the proliferation rate and differentiation on the chondrocytes. We first derived a natural ECM sheet from 10-μm-thick frozen sections of porcine knee cartilages. We then cultured the chondrocytes derived from a rabbit's knee on a dish precoated with the natural ECM. Then we assessed differentiation and chondrogenic potential of the cells compared with those grown in untreated culture dishes. We characterized the gene expression of chondrogenic markers, such as collagen type II, SOX-9, and aggrecan, as well as the level of ECM protein with the use of reverse-transcription polymerase chain reaction analysis. The cells cultured with the ECM sheet showed highest chondrogenic potential and differentiation. Therefore, we can induce good chondrogenesis by with the use of a natural ECM sheet on the culture dish. The readily available and easy-to-handle thin ECM sheets create an environment that promotes efficient cartilage regeneration. Our data suggest that this natural ECM scaffold improved the chondrogenic differentiation of the cells in vitro by providing a favorable microenvironment.

Highly Aligned Nanofibrous Scaffold Derived from Decellularized Human Fibroblasts.

Native tissues are endowed with a highly organized nanofibrous extracellular matrix (ECM) that directs cellular distribution and function. The objective of this study is to create a purely natural, uniform, and highly aligned nanofibrous ECM scaffold for potential tissue engineering applications. Synthetic nanogratings (130 nm in depth) were used to direct the growth of human dermal fibroblasts for up to 8 weeks, resulting in a uniform 70 μm-thick fibroblast cell sheet with highly aligned cells and ECM nanofibers. A natural ECM scaffold with uniformly aligned nanofibers of 78 ± 9 nm in diameter was generated after removing the cellular components from the detached fibroblast sheet. The elastic modulus of the scaffold was well maintained after the decellularization process because of the preservation of elastin fibers. Reseeding human mesenchymal stem cells (hMSCs) showed the excellent capacity of the scaffold in directing and supporting cell alignment and proliferation along the underlying fibers. The scaffold's biocompatibility was further examined by an in vitro inflammation assay with seeded macrophages. The aligned ECM scaffold induced a significantly lower immune response compared to its unaligned counterpart, as detected by the pro-inflammatory cytokines secreted from macrophages. The aligned nanofibrous ECM scaffold holds great potential in engineering organized tissues.