Name: improved 3d hydrogel cultures of primary glial cells for in vitro modelling of neuroinflammation
Uploaded: 2017-12-08T15:00:00
Description: Watch this Scientific Journal Video about Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation at JoVE.com

The authors are grateful for financial support from NSERC, CFI, AIHS, Alberta Health Services, and the Davey Endowment for Brain Research.

The protocol for the brain tissue extraction from day 1 Sprague Dawley rat pups, euthanized by decapitation, was approved by the Animal Care and Use Committee at the University of Alberta.
1. Microglia and Astrocyte Isolations67,68
NOTE: All media for isolation and cell culture is preheated to 37 &#176;C in a water bath. Hank&#39;s balanced salt solution (HBSS) has 1% penicillin-streptomycin (PS). All Dulbecco...

Towards the goal of generating a 3D culture system to model glial bioreactivity and the glial scarring process, we have developed a system that can support primary cultured microglia, astrocytes and oligodendrocytes and enables robust characterization of cell morphology and cell-cell interactions. From the micrographs shown, the morphology of each cell type was distinctly different with 2D, 3D-HAMA, and 3D HAMA-Basal lamina mixture platforms. In the 2D system, morphology was distinctly biased along the plane of the surfa...

Inflammation of the central nervous system (CNS) has long been considered a hallmark of acute (e.g., ischemic stroke, traumatic brain and spinal cord injury) and chronic (e.g. Alzheimer&#39;s, Parkinson&#39;s, and Huntington&#39;s diseases) CNS injury, but is increasingly recognized as a causal contributor to neurodegenerative and neuropsychiatric disorders. Sustained or inappropriate inflammation can cause neural injury and demyelination (e.g. multiple sclerosis), and negatively influence brain development (e.g., schizophrenia, autism) and mood states (e.g., depression, anxiety, bipolar disorder). Further, novel therapeutic strategies using implantable devices (e.g.,brain-computer-interfaces1,2,3, deep brain stimulation4,5, intraspinal microstimulation6,7,8,9,10) generate a predictable inflammatory response at the interface between the device and the CNS resulting in a protective tissue response that can cause loss of efficacy or device failure over the lifetime of the implant11. Inflammation in the CNS is typically initiated by microglia, which function as the resident immune cells of the CNS responsible for tissue surveillance and mounting the foreign body response (reviewed12). Depending on the severity of an insult, the microglia signal and recruit additional cell types to an injury site. Specifically, the microglia activate the astrocytes, which in turn act as secondary inflammatory cells and form a dense protective barrier to contain an injury site13,14. Microglia can also initiate an activity cascade in the cells of the peripheral immune system, which can result in the breakdown of the BBB to permit immune infiltration (reviewed in reference15).
In the case of devices implanted into the CNS, tissue damage resulting from device insertion as well as the continued presence of the foreign device may initiate a process termed glial scarring. In this process, the microglia migrate to and proliferate at the site of injury. They also initiate the release of inflammatory factors to neutralize potential threats and recruit additional glial cells. Subsequently, activated astrocytes become hypertrophic and begin encapsulating the implanted device to form a continuous fibrous barrier16. Inflammatory signalling also serves to promote withdrawal of neuronal processes from the vicinity of the implant and eventually recruits fibroblasts to reinforce the developing glial scar17. The oligodendrocytes, responsible for sheathing neurons in myelin to enhance conductance, do not survive this process and distant cells are partitioned from the implant by the scar18. Glial scarring greatly reduces the function and lifetime of implanted devices, particularly for recording electrodes, and ultimately serves to limit the functionality of neural interfaces19.
Several approaches have been exploited to increase the biocompatibility and interface activity of implanted devices in the CNS20,21,22,23. An extensive review is available on the biocompatible design of these neural interfaces24.The most prominent strategies include surrounding the electrode with compatible coatings such as polyelthyleneglycol (PEG), polylactic-co-glycolic acids (PLGA)25, or enhancing the electrode with conductive polymers such as poly(ethylene dioxythiophene) (PEDOT), and polypyrrole (PPy)26,27,28,29,30,31. Bioactive coatings have also been employed to provide cues for neural tissue growth using ligands derived from extracellular matrices including collagens, fibronectins, and hyaluronic acids32,33,34,35,36,37. The bioactivity of these coatings has been further explored with growth factor release systems to emulate natural cell secretions30,38,39,40,41,42,43,44,45,46,47,48,49,50. Simultaneously, some research groups have opted to remodel the electrode geometry, flexibility, and composition to decrease the mechanical mismatch between device and tissue51,52,53,54,55,56,57. Altogether, these strategies have lead to many promising improvements in next-generation neural interfacial devices, however the long-term compatibility is an on-going issue and progress may be hampered by complex and time-consuming in vivo models.
Animal model-based approaches can limit the experimental throughput and increase costs of testing electrode biocompatibility. In vitro approaches using conventional cell culture techniques offer a more cost-effective alternative but fail to recapitulate much of the complexity of the interaction between device and tissue58. In particular, testing of surface coatings using 2D cell culture limits the modeling of electrode geometry and the influence of mechanical mismatch and micromotion thought to contribute to generating a host response contributing to device failure59,60.
To overcome problems associated with 2D cell culture, hydrogel cultures of neural cells have been developed for a wide variety of applications, pharmacological studies61, to direct neural cell differentiation62, to understand disease pathways63,64, or layered in co-culture with other cell types to model cell migration, neuroprotection, or to model tissue microenvironments61. Hydrogels are readily formed at different sizes and geometries can incorporate numerous types of primary or immortalized cell cultures, and are highly amenable to analysis by commonly used techniques such as confocal fluorescence microscopy. To create a model of the glial scarring process, we have recently developed and characterised a hyaluronic-acid based 3D hydrogel system for high-throughput testing of the glial response to implanted electrodes (Figure 1)65. This system has several distinct advantages: 1) primary glial cells (microglia, astrocytes, and oligodendrocytes) are encapsulated in a 3D matrix composed of polymers of hyaluronic acid, which is an endogenous extracellular matrix component; 2) the matrix stiffness can be &#39;tuned&#39; to recreate the mechanical properties of brain or spinal cord tissue; and 3) cells can be encapsulated in the matrix in a rapid bench-top approach using photopolymerization with green light, limiting toxicity during encapsulation. This system enables key features of in vivo biocompatibility: devices are inserted into the hydrogel in a comparable manner to tissue, and the cellular response to implanted devices are monitored for a wide range of parameters65. These include mechanical mismatch between devices and the hydrogel coatings of various structures and electrical stimulation pulses. This system also includes oligodendrocyte and related precursors, which are often present and recruited in glial scars. Their damage, death, and phagocytosis by microglia are highly indicative of inflammatory injury and as a model reduced scarring or recovery, they have the capacity to demonstrate re-myelination of neurons66.
Herein we describe a method for synthesis and formation of hybrid hyaluronic acid hydrogels combined with commercially available basement membrane formulations to improve cell incorporation. Further, we will demonstrate the incorporation of primary cultured glial cells (microglia, astrocytes, and oligodendrocytes) and analysis of culture growth using immunocytochemistry and confocal microscopy.

<table>
	<tbody>
		<tr>
			<td>1. Materials for HAMA synthesis and photopolymerization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronic acid (HA)</td>
			<td>Sigma-Aldrich</td>
			<td>53747-10G</td>
			<td>Streptococcus equi, MW: 1.5 - 1.8 X 10^6</td>
		</tr>
		<tr>
			<td>Methacrylic anhydride (MA)</td>
			<td>Sigma-Aldrich</td>
			<td>275585-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>221465-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH)</td>
			<td>Commerical Alcohols Inc.</td>
			<td></td>
			<td>Anhydrous</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (pH 7.4) tablets</td>
			<td>Fisher Scientific</td>
			<td>18912014</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine (TEA)</td>
			<td>Sigma-Aldrich</td>
			<td>90279-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>1-Vinyl-2pyrrolidinone (NVP)</td>
			<td>Sigma-Aldrich</td>
			<td>V3409-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>EosinY (EY)</td>
			<td>Sigma-Aldrich</td>
			<td>E6003-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-(Trimethoxysilyl)propyl methacrylate</td>
			<td>Sigma-Aldrich</td>
			<td>440159-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker (100 mL)</td>
			<td>Corning</td>
			<td>1000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker (500 mL)</td>
			<td>Corning</td>
			<td>1000-600</td>
			<td></td>
		</tr>
		<tr>
			<td>pH paper (Labstick)</td>
			<td>Sigma-Aldrich</td>
			<td>9580</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>2. Materials for glial cell isolation and cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>P1-2 Sprague Dawley rat pups</td>
			<td>Charles River</td>
			<td>CD Sprague Dawley rat strain code 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissector scissors - slim blades (small)</td>
			<td>Fine Science Tools</td>
			<td>14081-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors - Toughcut (large)</td>
			<td>Fine Science Tools</td>
			<td>14130-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps (Dumont #5)</td>
			<td>Fine Science Tools</td>
			<td>11521-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved fine forceps (Dumont #7)</td>
			<td>Fine Science Tools</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14170-112</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium and Ham&#39;s nutrient mixture F-12 (DMEM/F12)</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (PS)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Gibco</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (PLL)</td>
			<td>Sigma-Aldrich</td>
			<td>P-6282</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>05-539-5</td>
			<td></td>
		</tr>
		<tr>
			<td>12 well Tissue culture treated plates (Cellstar)</td>
			<td>Greiner Bio-One</td>
			<td>665 108</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL serological pipette</td>
			<td>Fisher Scientific</td>
			<td>13-676-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL serological pipette</td>
			<td>Fisher Scientific</td>
			<td>12-676-10K</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (60 mm X 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (100 mm X 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverslip (18 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-100 18CIR</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>3. Materials for microscopy (confocal and scanning electron microscopy)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-CNPase</td>
			<td>abcam</td>
			<td>ab6319</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-Iba1</td>
			<td>Wako Laboratory Chemicals</td>
			<td>019-17741</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-GFAP</td>
			<td>abcam</td>
			<td>ab4674</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Fisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Fisher Scientific</td>
			<td>00-4958-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td>Buffered (10%)</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electon Microscopy Sciences</td>
			<td>157-8</td>
			<td>Buffered (8%)</td>
		</tr>
		<tr>
			<td>Guteraldehyde</td>
			<td>Electon Microscopy Sciences</td>
			<td>16019</td>
			<td>Buffered (8%)</td>
		</tr>
		<tr>
			<td>Osmium tetraoxide</td>
			<td>Electon Microscopy Sciences</td>
			<td>19152</td>
			<td>Buffered (2%)</td>
		</tr>
		<tr>
			<td>Hexamethyldilazane (HMDS)</td>
			<td>Electon Microscopy Sciences</td>
			<td>16700</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH)</td>
			<td>Electon Microscopy Sciences</td>
			<td>15055</td>
			<td>Anhydrous</td>
		</tr>
		<tr>
			<td>Microscope Slide (25 X 75 X 1 mm)</td>
			<td>VWR International</td>
			<td>48311-703</td>
			<td></td>
		</tr>
	</tbody>
</table>

improved 3d hydrogel cultures of primary glial cells for in vitro modelling of neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

To model the neural tissue host response and the glial scar in a high throughput, in vitro system requires a 3D cell scaffold with a matrix material that is biocompatible, does not incur a cytotoxic event during in situ formation, and is modifiable with bioactive components to guide benevolent response. To this end, we have created a 3D cell scaffold system based on hyaluronic acid, and encapsulated a primary mixed glial cell population to study cell-cell interactions an...

Watch this Scientific Journal Video about Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation at JoVE.com

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

The overall goal of this 3D cell culture protocol is to model the glial scar event of the central nervous system using key indicator cells:microglia, astrocytes and oligodendrocytes.This method can help answer key questions in the neuro-inflammation field, such as the potential for glial cells to enhance regenerative strategies and unravel the glial scar.The main advantage of this technique is that glial biology can be rapidly characterized in a 3D micro-environment with systematic and high-through put manipulations.To prepare cover slips for seeding, begin by adding one milliliter of 3-propyl methacrylate to 49 milliliters of distilled water to make a 2%solution.Then place 18 millimeter glass cover slips into the solution and rock for one hour at room temperature.After one hour, rinse the cover slips by serially dipping each cover slip in three beakers of 100 milliliters of deionized water.Then dry the cover slips in a vacuum with a desiccator and oven at 40 degrees Celsius overnight.The next day, use forceps to quickly immerse individual cover slips in 70%ethanol.Then, without drying, drop each cover slip into a well of a 12-well plate.Next, wash each well with one milliliter of sterile deionized water.Then add one milliliter of sterile poly

improved-3d-hydrogel-cultures-primary-glial-cells-for-vitro-modelling

Research

JoVE Journal

Bioengineering

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...