The authors would like to acknowledge NSERC Discovery Grant 402462 "Tissue engineered scaffolds for controlling induced pluripotent stem cell behavior".

Notes before starting protocol: Fibrinogen is a blood derived protein and thus appropriate safety training must be completed before handling. This protocol requires 2 days to complete so time the desired stem cultures appropriately to ensure they are ready for seeding. In terms of calculating how much fibrinogen to weigh out, three 35 mm petri dishes of tris buffered saline (TBS, pH 7.4) containing 110-130 mg of fibrinogen dissolved in 3 mL of TBS will be sufficient to produce 1 24 well plate containing 400 &mu;l fibrin scaffolds in each well.
1. Day One: Making fibrinogen solutions and overnight dialysis
<ol>
	<li>Take lyophilized fibrinogen out of the refrigerator and let it sit for 20 minutes to allow it to come to room temperature.</li>
	<li>Add 3 mL of Tris buffered saline (TBS) to each 35 mm petri dish. Each plate of fibrinogen yields 2 to 3 mL of fibrinogen solution ranging in concentration from 12 to 20 mg/mL and the total amount of fibrinogen needed can be calculated based on these approximations.</li>
	<li>Weigh out approximately 100-130 mg fibrinogen (Fg) and sprinkle onto the surface of the TBS present in each dish. Wait 5 minutes for the fibrinogen to begin to go into solution. Cover petri dish with lid.</li>
	<li>Incubate petri dishes at 37&deg;C for 2 hours to allow the fibrinogen to fully go into the TBS solution.</li>
	<li>Wet dialysis tubing (7000 MW cut off) with TBS. Fold bottom end of the tubing and clamp end shut with a dialysis clamp.</li>
	<li>Pipette the fibrinogen solutions from dishes into dialysis tubing. Fold over top end and clamp shut with a dialysis clamp.</li>
	<li>Place dialysis tubing containing fibrinogen solution into a 4 liter container of TBS. Mix solution using stir plate set to low speed (100 rpm).</li>
	<li>Dialyze solution overnight on stir plate for at least 12 hours. The TBS solution does not need to be changed over this time period.</li>
</ol>
2. Day 2: Polymerization of fibrin scaffolds and seeding of stem cells into scaffolds
All steps described for this process should be performed in a sterile tissue culture hood as these scaffolds will be seeded with stem cell cultures.
<ol>
	<li>Remove the fibrinogen solution from dialysis tubing and place into an appropriate sized conical tube (either 15 mL or 50 mL).</li>
	<li>Filter fibrinogen solution with a 5.0 &mu;m syringe filter to remove large impurities in the solution. Depending on the volume of the solution, the use of multiple filters may be necessary to process the entire solution.</li>
	<li>Sterile filter the fibrinogen solution with a 0.22 &mu;m filter into a 50mL conical tube. Using an appropriate size pipette, determine the volume of the filtered fibrinogen solution to use later when doing calculations. Depending on the volume of the solution, the use of multiple filters may be necessary to process the entire solution.</li>
	<li>Measure absorbance of the fibrinogen solution at 280 nm using the spectrophotometer. A dilution factor of 50 is often necessary to obtain an absorbance under 1.</li>
	<li>Calculate the concentration of protein present in the fibrinogen solution using the following equation: [Fibrinogen in mg/mL] = (A280 &times; Dilution Factor) &divide; 1.55 where 1.55 is the extinction coefficient at A280 for human fibrinogen 13. Next calculate the amount of TBS needed to dilute the concentration of the solution to 11.1 mg/mL of fibrinogen based on the initial volume. The final concentration of protein in the fibrin scaffolds will be 10 mg/mL after polymerization.</li>
	<li>Obtain sterile thrombin and calcium chloride solutions. First, add 15 &mu;L thrombin solution (concentration: 40 U/mL - final concentration in scaffold will be 2 U/mL) to right side of each well in the first row of the 24 well plate. Next, add 15 &mu;L of 50 mM calcium chloride solution to left side of each well. Finally, add 270 &mu;L of the fibrinogen solution to the plate. Shake plate from side to side followed by shaking back to front to ensure mixing of solutions.</li>
	<li>Let the row containing the mixtures polymerize for 5 minutes at room temperature. The scaffolds become more opaque as they polymerize.</li>
	<li>Repeat steps 2.6 and 2.7 until the desired number of fibrin scaffolds have been polymerized.</li>
	<li>After the last 5 minute incubation, place the plates inside of a 37&deg;C incubator to allow for complete polymerization of scaffolds.</li>
	<li>After the one hour incubation is complete, the next step is to seed the scaffold with embryoid bodies. Using a 20 &mu;L pipettemen, select an individual embryoid body and place in the center of the fibrin scaffold. Check with microscope that each well contains a single embryoid body.</li>
	<li>Add 5 &mu;L of thrombin solution (concentration: 40 U/mL) and 5 &mu;L of 50 mM calcium chloride solution on top of each embryoid body followed by 90 &mu;L of fibrinogen solution to each well. The solution should form a bubble encapsulating the embryoid bodies.</li>
	<li>Place plate back in 37&deg;C incubator for an additional hour to ensure polymerization.</li>
	<li>After incubation, add 1 mL of the appropriate cell culture media to each well and place plate back into 37&deg;C incubator.</li>
</ol>
3. Representative Results
Figure 1 shows a schematic of the side view of an individual well containing the 3D fibrin scaffold culture system seeded with an embryoid body. Figure 2A shows representative images of mouse induced pluripotent stem cells being cultured on mouse embryonic fibroblast feeder layers. These cells are then induced to form embryoid bodies, aggregates of cells containing neural progenitors, using an 8 day retinoic acid treatment protocol (Figure 2B) as previously described 14 while Figure 3 shows the appearance of an iPS-derived embryoid body after 3 days of culture inside of 3D fibrin scaffolds. Similar results have been obtained previously using mouse embryonic stem cells 7. Additionally, this method has been used to culture iPS-derived embryoid bodies produced using a different protocol involving retinoic acid and purmorphamine 15 inside of 3D fibrin scaffolds, demonstrating the versatility of this culture system.
<img alt="Figure 1." src="/files/ftp_upload/3641/3641fig1.jpg" /> 
Figure 1. Schematic showing the side view of an individual well containing a 3D fibrin scaffold seeded with an embryoid body.
<img alt="Figure 2." src="/files/ftp_upload/3641/3641fig2.jpg" /> 
Figure 2. Mouse induced pluripotent stem (iPS) cell culture and differentiation . To differentiate these cells into neural phenotypes, the iPS cells are cultured in suspension to produce aggregates of cells called embryoid bodies (EBs). These EBs are then treated with retinoic acid to induce neural differentiation and this protocol was previously used to induce neural differentiation of embryonic stem cells. A) Undifferentiated mouse iPS cell colony cultured on a mouse embryonic fibroblast feeder layer. B) Embryoid bodies derived from mouse iPS cells in suspension culture taken on day 8 after treatment with retinoic acid to induce neural differentiation. Scale bar is 100 &mu;m.
<img alt="Figure 3." src="/files/ftp_upload/3641/3641fig3.jpg" /> 
Figure 3. Example results of a mouse iPS embryoid body after 3 days of culture inside of a 3D fibrin scaffold. Cells have begun to migrate and differentiate inside of the fibrin scaffold. Scale bar is 500 &mu;m.

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The authors have nothing to disclose.

This protocol detailed above provides a method for generating 3D fibrin scaffolds for pluripotent stem cell culture, specifically for mouse embryonic and induced pluripotent stem cells. This 3D biomaterial based culture system more accurately mimics the stem cell niche found in vivo and as a result, it can be used to screen biological cues to determine their effects on stem cell differentiation 6. Our observations have shown that these scaffolds when seeded with stem cell derived embryoid bodies remai...

Equipment needed:
	Analytical balance 
pH meter 
Tissue culture incubator (37&deg;C, 5% CO2) 
Stir plate 
Spectrophotometer 
Sterile tissue culture hood
	Tris buffered saline (pH 7.4) (Need 4 L plus enough for dissolving fibrinogen) 
50 mM calcium chloride solution 
Sterile conical tubes (15 or 50 mL) 
35 mm Petri dishes 
Dialysis tubing (7,000 MW cutoff) 
Dialysis clips 
5.0 &mu;m syringe filters 
Individually wrapped sterile 0.22 &mu;m syringe filters 
Syringe 
24 well tissue culture plates

 <table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Fibrinogen (human)</td>
 <td>Calbiochem</td>
 <td>341578 </td>
 <td></td>
 </tr>
 <tr>
 <td>Thrombin (human)</td>
 <td>Sigma</td>
 <td>T7009</td>
 <td></td>
 </tr>
	</tbody>
 </table>

preparation of 3d fibrin scaffolds for stem cell culture applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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25.1K Views.  University of Victoria. This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Video: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

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 ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

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 ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...