The authors gratefully acknowledge funding support from the University of Cincinnati. The authors also thank Ron Flenniken of the University of Cincinnati Advanced Materials Characterization laboratory for support.

No potential conflict of interest was reported by the authors.

SCs can promote nerve regeneration due to their phenotypic transformation and regenerative potential following nerve injury. However, how ECM cues regulate this regenerative capacity remains mostly unclear, potentially hindering not only the development of biomaterials that aim to promote nerve regeneration but also the understanding of the mechanisms involved in nerve regeneration. To begin to examine this interplay, cell culture substrates were created where ECM cues such as stiffness, protein coating, and adhesive top...

Peripheral nervous system (PNS) injuries remain a major clinical challenge in healthcare by compromising the quality of life for patients and creating a significant impact through a multitude of socioeconomic factors1,2. Schwann cells (SC), as the major glial cells in the PNS, provide necessary molecular and physical cues to induce PNS regeneration and aid in functional recoveries in short gap injuries. This is due to the remarkable ability of SCs to dedifferentiate into a &#8220;repair&#8221; cell phenotype from a myelinating or Remak phenotype3. The repair SC is a distinctive cell phenotype in several ways. Following injury, SCs increase their proliferation rate by re-entering the cell cycle and begin expression of several transcriptional factors to facilitate reinnervation. These factors, such as c-Jun and p75 NTR, are upregulated while myelinating SC markers, such as myelin basic protein (MBP), are downregulated4,5. In addition, SCs change morphology to become elongated and aligned with each other to form B&#252;ngner bands across the injury site6. This provides a physical guidance mechanism for the axons to extend to the correct distal target7. However, despite the ability that SCs possess to promote nerve regeneration in short gap injuries, the outcome of functional recovery remains poor in severe injuries. This is due in part to a loss of extracellular matrix (ECM) guidance cues, as well as the inability of SCs to maintain the regenerative phenotype over long periods of time8.
The nerve regeneration and recovery process are intimately tied to the state of the basal lamina following injury. The basal lamina is a layer of ECM around the nerve that facilitates guidance and provides ECM-bound cues for axons and SCs in cases where it remains intact following injury9. The state of the ECM and its ability to deliver matrix bound cues to cells is vitally important and has been previously explored in a variety of different contexts10,11,12,13,14. For example, it has been shown that the stiffness of the ECM can guide cell functions such as proliferation and differentiation11,15,16. Composition of the ECM can also lead to a distinct cellular response and regulate cell behaviors such as migration and differentiation through intracellular signaling pathways17,18. Furthermore, cell morphology, including spreading area and cellular elongation, play a major role in regulating the function and can be governed by ECM-bound cues19,20. Many previous studies have focused on stem cells differentiating into defined lineages, yet SCs possess a similar ability to alter phenotype from a homeostatic, adult SC within a healthy nerve, to a repair SC capable of secreting proteins and growth factors while remodeling the ECM following nerve injury5,21. Therefore, it is especially crucial to identify mechanisms underlying the relationship between the innate SC regenerative capacity and ECM bound cues for the insight to ultimately harness this capacity for nerve regeneration.
To address this, we have developed a detailed methodology to produce a cell culture substrate where mechanical stiffness and ligand type can be easily tuned in physiologically relevant ranges. Polydimethyl siloxane (PDMS) was chosen as a substrate due to its highly tunable mechanics as compared to polyacrylamide gel, where the maximum Young&#8217;s modulus is around 12 kPa contrasted to PDMS at around 1000 kPa22,23,24. This is beneficial to the work at hand, as recent studies have shown the Young&#8217;s modulus of a rabbit sciatic nerve can exceed 50 kPa during development, thereby suggesting that the range of stiffness of nerves within the PNS is wider than previously examined. Different proteins are capable of adsorption onto PDMS substrates to analyze the combinatorial regulation of mechanics and ligands on SC behavior. This allows for the investigation of multiple microenvironmental cues present in the PNS regeneration process and comparison of a high degree of tunability to the work focusing solely on stiffness of the substrate25. Further, these engineered cell culture substrates are compatible with a multitude of quantitative analysis methods such as immunohistochemistry, western blot, and quantitative polymerase chain reaction (q-PCR).
This engineered cell culture platform is highly suitable for analyzing mechanistic pathways due to the high level of individual tunability of each ECM-bound signal. In addition, popular methods for cell micropatterning, including microcontact printing, can be achieved on the substrates to allow for controlled cellular adhesion to analyze cell shape in relation to other ECM bound cues24. This is critical because line patterned substrates, which promote elongation in cell populations, provide a tool to mimic and study elongated and regenerative SCs within B&#252;ngner bands during nerve regeneration. Further, cellular morphology is a potent regulator of multiple cell functions and can potentially introduce confounding experimental results if not controlled26,27. Significant attention is now being provided to the mechanisms governing the SC regenerative phenotype as regulated by ECM cues28,29,30. This is essential to provide insight into the design of biomaterials that can be applied as nerve guidance conduits for aid in PNS nerve regeneration. These detailed protocols can ultimately be applied as a potential tool to decipher the mechanisms of SC and other cell type function as regulated by ECM bound cues.

<table>
	<tbody>
		<tr>
			<td>Albumin from Bovine Serum (BSA), Texas Red conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>A23017</td>
			<td>BSA staining to show micropatterns</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7076S</td>
			<td>Antibody used for western blot analysis</td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7074S</td>
			<td>Antibody used for western blot analysis</td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Thermo Fisher Scientific</td>
			<td>B23151</td>
			<td>Reagent used to measure cell proliferation</td>
		</tr>
		<tr>
			<td>BrdU primary antibody conjugated with Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>B35130</td>
			<td>Used to visualize BrdU in cell proliferation assays</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10483-01</td>
			<td>Protein used to coat coverslips</td>
		</tr>
		<tr>
			<td>Compression force test machine</td>
			<td>TestResources</td>
			<td></td>
			<td>Instrument to quantify mechanical properties of polymers</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000044</td>
			<td>Cell culture medium supplemental</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermo Fisher Scientific</td>
			<td>33010-018</td>
			<td>Protein used to coat coverslips</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti2</td>
			<td>Fluorescence microscope</td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>78440</td>
			<td>Protease and Phosphatase Inhibitor</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Thermo Fisher Scientific</td>
			<td>23017015</td>
			<td>Protein used to coat coverslips</td>
		</tr>
		<tr>
			<td>Mounting medium with DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36971</td>
			<td>Coverslip mountant and nuclei staining</td>
		</tr>
		<tr>
			<td>Mouse c-Jun primary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>711202</td>
			<td>Primary antibody to visualize c-Jun protein</td>
		</tr>
		<tr>
			<td>Mouse &#946;-Actin primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3700S</td>
			<td>Loading control for western blot experiments</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Cell culture medium supplemental</td>
		</tr>
		<tr>
			<td>Photoresist SU 2010</td>
			<td>KAYAKU</td>
			<td>SU8-2010</td>
			<td>Photoresist</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma Aldrich</td>
			<td>P-2443</td>
			<td>Block non-specific protein binding</td>
		</tr>
		<tr>
			<td>Rabbit c-Jun primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9165S</td>
			<td>Primary antibody for visualization of c-Jun protein</td>
		</tr>
		<tr>
			<td>Rabbit myelin basic protein primary antibody</td>
			<td>Abcam</td>
			<td>ab40390</td>
			<td>Primary antibody for visualization of MBP</td>
		</tr>
		<tr>
			<td>Rabbit p75NTR primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>8238S</td>
			<td>Primary antibody for visualization of p75NTR</td>
		</tr>
		<tr>
			<td>Rhodamine phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>R415</td>
			<td>Visualization of cell cytoskeleton</td>
		</tr>
		<tr>
			<td>RIPA buffer</td>
			<td>Abcam</td>
			<td>ab156034</td>
			<td>Cell lysis buffer</td>
		</tr>
		<tr>
			<td>RT4-D6P2T Schwann cell line</td>
			<td>ATCC</td>
			<td>CRL-2768</td>
			<td>Cell line used in experiments</td>
		</tr>
		<tr>
			<td>SYLGARD 184 PDMS base and curing agent</td>
			<td>Sigma Aldrich</td>
			<td>761036</td>
			<td>Tunable polymer used to coat coverslips</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15090-046</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr>
			<td>UV-Ozone cleaner</td>
			<td>Novascan</td>
			<td></td>
			<td>Increase hydrophicility of PDMS</td>
		</tr>
		<tr>
			<td>Versene (1x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15040066</td>
			<td>Cell dissociation reagent</td>
		</tr>
	</tbody>
</table>

preparation of tunable extracellular matrix microenvironments to evaluate schwann cell phenotype specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

To analyze and quantify the interplay between substrate stiffness and protein composition on SC phenotype, a tunable PDMS cell culture substrate was developed (Figure 1A). Compression testing of the polymer at differing base: curing agent ratios was utilized to quantify the Young&#8217;s modulus (E) of the substrate (Figure 1B). The resulting range of modulus values represents physiologically relevant substrate conditions. Following preparation of substrates, SC...

Watch this Scientific Journal Video about Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification at JoVE.com

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

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Bioengineering

5.2K Views.  University of Cincinnati. This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Video: Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...