The authors would like to thank Mr Kajen Suresparan, Dr Jonathan Wubetu, Mr Dominik Grudzinski, Miss Chen Zhao, and Dr Thierry Pilot for their help in the optimization of alginate microbead fabrication, cell culture and A&#946; detection, and useful scientific discussions.

1. Preparations
<ol>
	<li>Prepare 100 mL of ~4% (w/v) alginate stock solution.
	<ol>
		<li>Weigh 4.4 g of alginate sodium salt and add the dry powder to 100 mL of HEPES-buffered saline (HBS, 20 mM HEPES (0.477 g) with 150 mM NaCl (0.877 g) in deionized water allowing hydration.</li>
		<li>Use a magnetic stirrer at ~500 revolutions per minute (rpm) and heat to up to 50 &#176;C to allow complete alginate hydration. 
		NOTE: The alginate may need one or two hours to be completely hydrated. To save on time, alginate solution can be made up a day in advance of the encapsulation protocol and stored at 4 &#176;C until the next day. When alginate has been refrigerated, heat gently to 37 &#176;C using a water bath or a hot plate and agitate with a magnetic stirrer immediately before use.</li>
		<li>Sterile filter the alginate solution using a 0.22 &#181;m pore polyethersulfone (PES) filter before use. Filtering is recommended at 37 &#176;C to allow easy flow of alginate. The viscosity will increase if the temperature is allowed to drop.</li>
	</ol>
	</li>
	<li>Prepare 1,000 mL of 0.5 M CaCl2.
	<ol>
		<li>Weigh 55.49 g of anhydrous CaCl2 and dissolve in 1,000 mL of HBS (20 mM HEPES and 150 mM NaCl in 1,000 mL of deionized water).</li>
		<li>Sterile filter using a 0.22 &#181;m pore PES filter.</li>
	</ol>
	</li>
	<li>Prepare 100 mL of dissolution mix.
	<ol>
		<li>Prepare a 100 mM HEPES (23.83 g) and 500 mM trisodium citrate dihydrate (147.05 g) solution in phosphate-buffered saline.</li>
		<li>Adjust to pH 7.4 using NaOH or HCl.</li>
	</ol>
	</li>
</ol>
2. Setting up the encapsulation system
<ol>
	<li>Clean the laminar flow hood.
	<ol>
		<li>Sterilize the laminar flow hood containing the encapsulator using UV exposure followed by spraying with 70% v/v ethanol solution.</li>
		<li>Flush the encapsulator system with 10 mL of 70% v/v ethanol solution followed by 10 mL of sterile deionized water.</li>
		<li>Attach the 300 &#181;m nozzle and repeat step 2.1.2.</li>
		<li>Spray the bottle containing the filtered alginate solution, the bottle containing filtered calcium chloride solution and any tools, equipment and empty culture plates that will be used with 70% v/v ethanol solution and place them into the laminar flow hood. Prior to use, switch on a UV light again for 30 min to thoroughly sterilize.</li>
		<li>Prepare a waste beaker filled with a biological grade disinfectant solution and place the beaker into the hood.</li>
		<li>Fill a beaker with 100 mL of sterile CaCl2 (aqueous) solution and add in a magnetic stirrer. Set the stirring speed to 100 rpm. Place the beaker on a magnetic platform at a height of 18 cm from the tip of the nozzle. This beaker will be used to collect alginate beads and allow their gelation.</li>
	</ol>
	</li>
	<li>Prepare cells for encapsulation.
	<ol>
		<li>Remove cells from the incubator and detach from the near-confluent flask using 0.25% trypsin-EDTA solution and incubate at 37 &#176;C for 5-10 min.</li>
		<li>Isolate a sample for estimation of cell density and then centrifuge the remaining cells at 1,000 rpm for 5 min to obtain a cell pellet.</li>
		<li>Resuspend the pellet in HBS (20 mM HEPES and 150 mM NaCl) to double the final desired cell concentration (e.g., for a final concentration of 1.5 x 106 cells/mL, resuspend the cells in HBS to achieve a concentration of 3 x 106 cells/mL).</li>
		<li>In a 50 mL centrifuge tube, mix the cell suspension in a 1:1 ratio with ~4% (w/v) alginate solution to obtain a final suspension containing the desired cell concentration (e.g., 1.5 x 106 cells/mL) in a ~2% (w/v) alginate solution.</li>
	</ol>
	</li>
	<li>Set the encapsulation parameters.
	<ol>
		<li>Set the speed of the encapsulator machine to the maximum extrusion speed (8.9 mL/min), the voltage to 1.0 kV and the frequency to 5,500 Hz. 
		NOTE: These parameters were previously optimized to obtain microbeads of 550 &#181;m in diameter.</li>
	</ol>
	</li>
</ol>
3. Fabrication
<ol>
	<li>Fabricate the microbeads.
	<ol>
		<li>In a 20 mL syringe, load 5 mL of the cell-alginate suspension and attach a syringe to the encapsulator.</li>
		<li>Start the encapsulator by activating the flow which will push the cell-alginate suspension through the feeder. A stream of droplets will be extruded through the nozzle.</li>
		<li>Collect the first 1 mL in the waste beaker to void the initial non-uniform stream.</li>
		<li>Continue to run the remaining 4 mL allowing the droplets to fall into the CaCl2 gelation bath. Each milliliter can be run separately (but successively) and collected in four different gelation baths if required. 
		NOTE: Upon contact with the gelation bath, the alginate in the droplets will instantly cross-link with the calcium ions in the gelation bath and form spherical microbeads.</li>
		<li>After one minute, remove the gelation beaker from the magnetic platform and allow the microbeads to sit for a further 4 min without agitation (time necessary to allow complete gelation across the microbeads at room temperature).</li>
	</ol>
	</li>
	<li>Retrieve microbeads.
	<ol>
		<li>Remove any large alginate debris or artefacts using a pair of sterile tweezers, and then use a sterile plastic pipette to transfer to a 74 &#181;m mesh filter to retrieve the microbeads from the gelation bath.</li>
		<li>Transfer the microbeads into a centrifuge tube using the appropriate culture medium and allow to equilibrate for 5 min in the cell culture medium.</li>
		<li>Transfer to a flask, plate or Petri dish for incubation and further experiments. 
		NOTE: Gelation bath should not be reused.</li>
	</ol>
	</li>
</ol>
4. Testing and use of microbeads
<ol>
	<li>Test microbead quality.
	<ol>
		<li>To assess the cell viability (or other biological readouts) after encapsulation and culture, gently disrupt the microbeads to release the encapsulated cells using the dissolution mix. 
		NOTE: The required volume of dissolution mix is dependent on the number of microbeads used per test. A suggested ration is 4 mL of dissolution mix to every 1 mL of encapsulated cells. This step only needs to be performed on a single sample fro each microbead population.</li>
		<li>Incubate the cells in a cell culture incubator supplemented with 5% CO2 at 37 &#176;C for 10 min.</li>
		<li>Estimate cell viability for cells now in solution as usual by staining cells with trypan blue and using a hemocytometer chamber. 
		NOTE: If using automated cell counters for estimations of cell viability, care should be taken to avoid alginate artefacts interfering with the counts. In those cases, naual cell counting is advised.</li>
		<li>To assess microbead stability, measure the average diameter for a sample from each microbead population over a time course using a microscope and imaging software. Dramatic subsequent variations in diameter can be indicative of alginate degradation.</li>
	</ol>
	</li>
	<li>Use microbeads for detection of secreted A&#946;.
	<ol>
		<li>Sample cell culture media from 7PA2 cells cultured in standard conditions (2D) at 24 h intervals and store at -20 &#176;C for further analysis.</li>
		<li>Sample cell culture media from encapsulated 7PA2 cells cultured in standard conditions (3D) at 24 h intervals and store at -20 &#176;C for further analysis.</li>
		<li>Detect secretion of A&#946; from 7PA2 cells into collected conditioned media by enzyme-linked immunosorbent assay (ELISA) (Figure 4).</li>
	</ol>
	</li>
	<li>Use the microbeads for further study in relevant applications. 
	NOTE: Encapsulated 7PA2 cells can be used to assess the effects of sustained A&#946; secretion in any or model.
	<ol>
		<li>For microbead engraftment within the rat hippocampus in brains, generate 1 mm thick coronal sections of the rat brain and use surgical tools to insert microbeads into the desired area (Figure 5).</li>
		<li>Encapsulate different cell types in alginate microbeads following the described protocols to assess the sustained release of the biomolecules of interest. Relevant in vitro and in vivo systems can be further modeled.</li>
		<li>Detect the expression of membrane-bound markers of encapsulated cells using flow cytometry and/or immunofluorescence. 
		NOTE: An example of the use of a similar method for the fabrication of microbeads modeling early stages of tumor mass growth and biomarker expression is described by Rios 20.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The method outlined in this article is useful for encapsulating cells achieving a narrow size distribution of alginate microbeads27. It also affords the advantage of growing cells in an immuno-isolated environment17,19, protecting them from external stress. Additionally, encapsulation of cells in alginate more closely mimics physiological conditions, specifically with regards to cell-to-cell interactions and matrix stiffness20. These factors are all particularly crucial for subsequent use in relevant applications, such as in vivo engraftment, precluding potential immune reactions in the surrounding tissue17. Furthermore, a major advantage of this protocol is the easy tuneability according to the application of interest: it is possible to modify the protocol and optimize the parameters for fabrication of larger or smaller, and softer or stiffer beads. The described method is used to fabricate beads small enough to be injected with minimally invasive methods, and sufficiently large enough to host a number of cells and provide a sufficient release of A&#946; to result in observable behavioral and pathological effects upon injection in animal models.
The success of this protocol is dependent on a number of critical steps. Careful cell handling and optimal cell culturing techniques are important to maintain cell viability and function20,28. Using standard culture conditions ensures the conservation of normal function of 7PA2 cells, as observed. This allows a similar release profile for A&#946;1-42 from encapsulated cells when compared with 2D cultures. In addition, for the protocol to work optimally, a low-viscosity alginate solution guarantees better results compared with a high viscosity alginate solution. This ensures that a homogenous laminar jet is extruded through the nozzle and an even distribution of cells within the matrix of the fabricated beads27. Materials used to encapsulate cells must have a very rapid gelation mechanism, allowing retention of shape.
Another critical step in this protocol is the handling of fabricated microbeads following gelation. Here, we show the retrieval of microbeads by using a large aperture plastic pipette to transfer the beads. Alternatively, pouring the calcium chloride solution containing the microbeads into a mesh filter can be used for bead retrieval. A large (5, 10 or 25 mL) serological pipette can be used to draw up the microbeads and then washed through the mesh filter instead of pouring. The advantage of this is a higher confidence in the sterility of the procedure compared to pouring. However, of the limitations is that some beads may be distorted if they are compressed by the pipette, in addition to risking a lower yield if a large proportion of microbeads are not rescued.
This approach has been used to encapsulate different cell lines to model and study different diseases (e.g., release of insulin from encapsulated pancreatic islet). The novelty of our approach is the engraftment of the microbeads generated using this protocol as a useful method in modeling important aspects of Alzheimer&#8217;s disease in vivo. When comparing the release profile of A&#946; from encapsulated cells (Figure 4) to the levels of A&#946; generated by a bolus injection (such as that reported in other studies3,26), a more chronic and sustained release of A&#946; can be expected. Figure 6 illustrates the predicted tendency that may be achieved. Using this system for in vivo modeling is more relevant to the way the disease progresses and can be more useful in drug discovery and development.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59597/59597fig6.jpg" /> 
Figure 6: Predicted release profile of A&#946;1-42 from encapsulated 7PA2 cells compared with a bolus injection. The profile of A&#946; release from engrafted 7PA2-containing microbeads allows the testing of the effects of chronic and sustained A&#946; in an animal model of relevance to AD. Conversely, a bolus injection would create a spike in A&#946; levels over a short period of time, followed by a rapid clearance of A&#946; from the brain. <a href="https://www.jove.com/files/ftp_upload/59597/59597fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Modeling neurodegenerative disease is challenging due to the complex and intricate nature of the brain. In Alzheimer&#8217;s disease (AD), the progressive loss of synaptic function and death of neurons is believed to be a downstream effect of sustained overproduction and accumulation of amyloid beta (A&#946;) peptides following abnormal processing of the amyloid precursor protein (APP) according to the amyloid cascade hypothesis1.
In order to understand the mechanisms of this amyloid-induced pathology and to aid in the identification of novel treatment targets, scientists have developed various in vivo preclinical models. One category of models utilizes a bolus injection of a synthetic A&#946; peptide into the rat brain2,3,4. The main limitation of such models is that they rely on a single-point or repeated treatments with A&#946; peptides in high concentrations deposited all in one go. This is inconsistent with the chronic, sustained nature of the release of A&#946; in disease5. Another category of in vivo models is transgenic animal models expressing one or more genetic mutations linked with the familial variations of the disease6,7,8,9,10. However, since familial AD only accounts for fewer than 5% of all Alzheimer&#8217;s cases11, the relevance of these models in translating to sporadic AD in humans is questionable12. Another drawback of the transgenic approach is the accelerated A&#946; formation from birth, which translates into deficits in cognitive function and pathological changes too quickly and aggressively to resemble disease progression in sporadic AD in patients12. For example, the 5x FAD model produces plaques in as little as 1.5 months13.
Interestingly, both these categories result in changes in cognitive function of relevance to AD research2,3,4,5,6, and sometimes they are accompanied by the appearance of pathological hallmarks of the disease such as amyloid plaques6,8, tau phosphorylation6,7 and/or synaptic and neuronal loss7,9,14. But while these types of models may give us an insight into the effects of high levels of amyloid in the brain, which are often associated with later stages of AD, they fail to reflect the earlier changes exhibited in response to the chronic and sustained exposure to the A&#946; peptide12, such as altered expression of synaptic markers15 and components in the extracellular matrix16. Therefore, there still remains a need for creating a chronic model that more accurately illustrates the effects of sustained A&#946; secretion on in vivo cognition and illustrates changes in pathology.
To this end, we have developed a system which allows the constant, sustained secretion of A&#946; in a controlled manner by immobilizing amyloid-secreting cells within hydrogel microbeads, which can be subsequently implanted within the adult rat brain to model aspects of sporadic AD.
Alginate was the selected biomaterial as it is biocompatible and does not induce any adverse responses when implanted in vivo17. Cell encapsulation in alginate hydrogels has been well established over the last four decades. The first example of its translation to the clinic was reported for the treatment of type 1 diabetes mellitus17. The earliest report of successful encapsulation of islets of Langerhans dates back to 1980. Transplantation of microbeads containing insulin-secreting cells revolutionized treatment options for diabetic patients as it restored pancreatic function, eliminating the need for insulin injection therapy18. These works report on how cell encapsulation can protect them from external stresses, whether mechanical or chemical. In fact, alginate beads act as a barrier and isolate cells from the surrounding environment preserving their phenotype, whilst allowing sufficient access to the surrounding media for nutrients and clearance of cellular byproducts19. Moreover, the use of alginate allows matching of mechanical properties of soft tissue20. Alginate hydrogels can be tuned to have a stiffness of 1-30 kPa, by simply varying alginate concentration and cross-linking density20,21. This is an essential aspect, not only to maintain the phenotypic expression of encapsulated cells in vitro, but also to avoid any inflammatory effects after engraftment in vivo22.
In this protocol, 7PA2 cells - a Chinese hamster ovary cell line that is stably transfected with a human APP V717F mutated gene23 -&#160;are used. These cells continuously produce catalytic products of APP, including A&#946;1-4224,25, and have been used to generate A&#946; as an alternative to synthetic production in preclinical, acute in vivo studies26. We describe a fabrication method for immobilizing 7PA2 cells within &#39;soft&#39;&#160;alginate microbeads, designed to allow the sustained secretion of biomolecules. As a proof of concept, we report on the release of the A&#946;1-42 peptide over time. The alginate that is used is a low-viscosity alginate with a molecular weight of 120,000-190,000 g/mol and a mannuronic to guluronic ratio of 1.56 (M/G).
In further studies, these microbeads can be safely transplanted within regions of the rat brain of relevance to AD (e.g., the hippocampus) to study the effects of chronic A&#946; secretion on behavior in vivo and pathology ex vivo. In addition, this system can be used to study the effects of chronic A&#946; release in in vitro and ex vivo applications. For instance, 7PA2-containing alginate microbeads can be co-cultured in vitro with neuronal or astrocytic cultures to assess the effects of chronic A&#946; exposure on cellular mechanisms associated with AD. Furthermore, this method can be used to examine the relationship between chronic A&#946; production and long-term potentiation in ex vivo electrophysiology.
The highlight of this protocol is the modularity and flexibility of the fabrication method, which enables the fabrication of alginate beads with a target dimension by fine tuning fabrication parameters. Depending on the application, the protocol can be adjusted to obtain bespoke targets with regards to microbead size, density of the encapsulated cells, and microbead stiffness. This protocol can be used for the encapsulation of a variety of cell types, developing more relevant three-dimensional (3D) in vitro models to study different pathologies. We recently reported on how alginate-encapsulated cells can be used to model early stages of cancer progression20.
The concept of the encapsulation process is based on the extrusion of a laminar jet of cells suspended in alginate solution through a nozzle. A vibrating head disrupts the jet with a controlled frequency resulting in equally sized alginate-based droplets. An external electric field allows separation of the formed alginate-based droplets, which upon contact with a solution enriched in divalent ions, such as calcium ions, can quickly cross-link, preserving their spherical shape. Incubation in the gelation solution allows formation of spherical microbeads containing cells within a homogeneous physical hydrogel27. Target size of microbeads and alginate hydrogels allows nutrient<s>s</s> and oxygen exchange with cell culture media for long periods of time (weeks). Figure 1A show a schematic representation of the encapsulation apparatus used (Figure 1B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59597/59597fig1.jpg" /> 
Figure 1: The encapsulation system. (A) Schematic representation of the encapsulation system. An alginate-cell suspension is loaded into a syringe (2) and fed through a reservoir at an extrusion speed set at syringe pump (1). In the reservoir, a vibration hat (3) vibrates at a frequency set by a waveform generator (4) to disrupt the stream at equal intervals, forming equally-sized droplets. As the solution is fed through a nozzle (5) and droplets are formed, an electrostatic potential is applied across an electrode (7) set by a voltage generator (6), which slightly charges the surface of the droplets, allowing the stream to spread as a result of repelling electrostatic forces. As droplets engage with the gelation bath (8), Ca2+-driven cross-linking of alginate results in the formation of spherical microbeads. (B) Photograph of the encapsulator before fabrication of alginate microbeads. <a href="https://www.jove.com/files/ftp_upload/59597/59597fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Microbead size can be altered depending on the intended use. In order to control the size of the microbead, the various parameters outlined in Figure 1A and in the protocol are adjusted accordingly. The internal diameter of the nozzle used has a substantial impact on the size of the droplets; further adjusting encapsulation parameters, namely extrusion speed, vibration frequency and voltage, is key to achieving a consistent size distribution. Table 1 outlines how the different parameters can alter the size of microbeads achieved with this system.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Parameter</td>
			<td>Nozzle size</td>
			<td>Vibration frequency</td>
			<td>Flow rate</td>
			<td>Electrode voltage</td>
		</tr>
		<tr>
			<td><img alt="Image 1" src="/files/ftp_upload/59597/59597img01.jpg" /></td>
			<td><img alt="Image 1" src="/files/ftp_upload/59597/59597img01.jpg" /></td>
			<td><img alt="Image 1" src="/files/ftp_upload/59597/59597img01.jpg" /></td>
			<td><img alt="Image 1" src="/files/ftp_upload/59597/59597img01.jpg" /></td>
		</tr>
		<tr>
			<td>Bead size</td>
			<td><img alt="Image 1" src="/files/ftp_upload/59597/59597img01.jpg" /><img alt="Image 1" src="/files/ftp_upload/59597/59597img01.jpg" /></td>
			<td><img alt="Image 2" src="/files/ftp_upload/59597/59597img02.jpg" /></td>
			<td><img alt="Image 1" src="/files/ftp_upload/59597/59597img01.jpg" /></td>
			<td>&#8211;</td>
		</tr>
	</tbody>
</table>
Table 1: Fabrication parameters and their influence on microbead size. The table illustrates how each parameter can influence the resultant size of fabricated microbeads, irrespective of the nozzle and the viscosity of the solution used.

<table border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="847">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">&#181;Manager software</td>
			<td style="width:261px;">Vale Lab, UCSF, USA</td>
			<td style="width:100px;">v.1.46</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">0.22 um PES filter</td>
			<td style="width:261px;">Merck, UK</td>
			<td style="width:100px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">15mm Netwell insert 74 um mesh filter</td>
			<td style="width:261px;">Constar, usa</td>
			<td style="width:100px;">#3477</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Alginic acid sodium salt from brown algae</td>
			<td style="width:261px;">Sigma-Aldrich,uk</td>
			<td style="width:100px;">A0682</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Calcium chloride</td>
			<td style="width:261px;">Sigma-Aldrich,uk</td>
			<td style="width:100px;">C1016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:341px;">CellTiter96&#160; AQueous One Solution cell proliferation assay</td>
			<td style="width:261px;">Promega, USA</td>
			<td style="width:100px;">G3580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Encapsulator</td>
			<td style="width:261px;">Inotech</td>
			<td style="width:100px;">IE-50</td>
			<td>serial no. 05.002.01-2005</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">HEPES</td>
			<td style="width:261px;">Sigma-Aldrich,uk</td>
			<td style="width:100px;">H4024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Hu A&#946; 1-42 ELISA</td>
			<td style="width:261px;">ThermoFisher, UK</td>
			<td style="width:100px;">KHB3441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">ImageJ software</td>
			<td style="width:261px;">ImageJ</td>
			<td style="width:100px;">v1.49p</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Inverted light microscope</td>
			<td style="width:261px;">Olympus</td>
			<td style="width:100px;">CKX41</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Leica microscope</td>
			<td style="width:261px;">Leica microsystems, UK</td>
			<td style="width:100px;">DMI6000B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Neo sCMOS Camera</td>
			<td style="width:261px;">Ander, UK</td>
			<td align="right" style="width:100px;">5.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Phosphate buffered saline</td>
			<td style="width:261px;">Sigma-Aldrich,uk</td>
			<td style="width:100px;">D1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Sodium chloride</td>
			<td style="width:261px;">Sigma-Aldrich,uk</td>
			<td align="right" style="width:100px;">433209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Trispdium citrate dihydrate</td>
			<td style="width:261px;">Sigma-Aldrich,uk</td>
			<td style="width:100px;">W302600-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:341px;">Trypsin-EDTA solution</td>
			<td style="width:261px;">Sigma-Aldrich,uk</td>
			<td style="width:100px;">T4049</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of amyloid-&#946;-secreting alginate microbeads for use in modelling alzheimer's disease

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic amyloid-&#946; (A&#946;) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic A&#946; production.
Chinese hamster ovary cells previously transfected with a human APP gene, secreting A&#946; (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of A&#946; was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 &#956;m for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted A&#946; were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h).
A concentration of 1.5 x 106 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 &#956;m, 3) containing about 100-150 cells per microbead and 4) able to secrete A&#946;.
In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.

7PA2 cells are successfully encapsulated in alginate microbeads 
After preparation, uniform and spherical alginate microbeads are successfully generated using this protocol. The images in Figure 2A below showcase an example of how altering one of the parameters (i.e., voltage) changes the flow and dispersion of the alginate droplets stream. Figure 2B shows an example of obtained alginate beads immediately after the gelation process.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59597/59597fig2.jpg" /> 
Figure 2: Fabrication method optimization. (A) Photographs showing changes in stream dispersion. (B) Bright-field image showing alginate spherical microbeads immediately after fabrication. (C) Example of optimization step: tuning selected fabrication parameters to achieve the target microbead size. Selected graphs to illustrate the relationship between each parameter and the size of the resultant microbeads (size distribution from at least n = 100 microbeads). Note that the nozzle internal diameter, viscosity of ejected solution and gelling conditions can also influence the size of fabricated beads. Error bars represent S.D. <a href="https://www.jove.com/files/ftp_upload/59597/59597fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The described protocol is flexible and allows the fabrication of different sizes of alginate-based microbeads, varying in composition according to the application. In Figure 2C, we report the influence of alginate flow rate, voltage and frequency on microbead size using a 2% (w/v) alginate solution in HEPES (pH 7.2) extruded through a 300 &#956;m nozzle.
As the scope of this study was to produce microbeads that can be embedded within the rat brain, we adjusted the fabrication parameters to obtain an average microbead diameter in the range of 500-600 &#956;m. For quality control purposes, the method was optimized to have minimal variability across the population of fabricated beads (i.e., narrow size distribution). Please note that (1) produced beads can be injected in the rat brain using a Hamilton syringe equipped with a 20G needle; and (2) one hemisphere of rat brain can host up to two or three beads of this size.
After optimization, encapsulating 7PA2 cells at a concentration of 1.5 x 106 cells/mL suspended in a 2% (w/v) alginate solution buffered with HEPES and dropped in a 0.5 M calcium chloride gelation bath yielded the desired microbeads. Figure 3A below shows encapsulated 7PA2 cells evenly distributed in microbeads after one day in standard cell culture conditions. 7PA2 cell proliferation was tested using an MTS assay as per manufacturer&#8217;s protocol. There was no significant difference between the behavior of 7PA2 cells grown with or without alginate over a seven-day period (Figure 3B). When incubating encapsulated cells in standard culture conditions, cells are expected to continue to proliferate over the duration of the experiment, and small degrees of cell escape can be expected as a result, as reported in other works28. The effects of this can be mitigated by encapsulating at a smaller cell density or reducing the serum concentration in which microbeads are incubated.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59597/59597fig3.jpg" /> 
Figure 3: Encapsulated 7PA2 cells. (A) Bright-field image of encapsulated 7PA2 cells showing even cell distribution throughout the alginate microbead. Fabrication parameters were set to obtain ~150 7PA2 cells per bead. No significant variation in alginate beads was observed over 7 days of culture. (B) There was no observed difference between the overall proliferation of 7PA2 cells incubated with alginate versus cells incubated without alginate. 7PA2 cells grow and migrate across the volume of the alginate beads; reduction of serum concentration can be considered to slow down cell growth. Error bars represent S.D. <a href="https://www.jove.com/files/ftp_upload/59597/59597fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Alginate microbeads encapsulating 7PA2 cells are stable over time 
To investigate the size and shape of obtained alginate microbeads, microscope analysis was performed after fabrication and gelation. The average diameter for the microbeads obtained using the selected protocol is 550 &#177; 2 &#956;m.
Theoretically, the number of cells expected in a 550 &#956;m-diameter microbead can be calculated as follows:
<img alt="Equation 1" src="/files/ftp_upload/59597/59597eq1.jpg" />
where V = volume and r = radius. The volume of a single microbead is V = 8.8 x 10-5 mL; therefore, the number of cells per microbead at time of encapsulation = (1.5 &#215; 106) x (8.8 x 10-5) &#8776; 130 cells.
Experimentally, we counted the number of encapsulated cells immediately after fabrication. Alginate beads were gently disrupted using the dissolution mix and cells were stained with trypan blue solution. Viability estimation and counting of cells was performed using a hemocytometer; obtained results showed an average of 116 &#177; 17 live cells per microbead (n = 5, data not reported). As expected, a minor difference between theoretical and experimental cell count immediately after encapsulation was observed. For some applications, and in particular to determine the amount of released A&#946; over time, it is important to predict the number of cells encapsulated in each alginate bead.
Interestingly, the encapsulation process does not have a significant impact on cell viability. Results are reported in a previous work, in which colorectal cancer cells (i.e., HCT-116) were encapsulated using a similar method, with no differences in cell viability in alginate microbeads compared to 2D controls20.
To measure the stability of the alginate used in the encapsulation process, we measured microbead diameters over a 14-day period (n = 100). There were no observed changes in average diameter 14 days after encapsulation as compared with immediately after encapsulation (data not reported).
Encapsulated 7PA2 cells release A&#946; over time 
Conditioned media analyzed from 2D and 3D cultures of 7PA2 cells reveal a constant increase in A&#946;1-42 levels. Cell culture media was sampled every 24 h, and up to four days, and analyzed using ELISA. Our data shows that the rate of release of A&#946;1-42 from microbeads (3D) is similar in profile to that released from the 2D culture (Figure 4).
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59597/59597fig4.jpg" /> 
Figure 4: Rate of A&#946;1-42 secretion from 7PA2 cells. (A) A&#946; release (% normalized after 4 days) from 2D and 3D in vitro models has a similar profile. Both models show a constant increase in A&#946; levels over the four-day period and, as expected, a steady concentration is not reached. (B) Concentration of A&#946;1-42 normalized to initial cell number, showing similar secretion of A&#946;1-42 over time, regardless of the culturing methods. Neither the presence of alginate nor the process of encapsulation (3D in vitro model) alter the secretion of A&#946;1-42. Error bars represent S.D. <a href="https://www.jove.com/files/ftp_upload/59597/59597fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Potential application of encapsulated 7PA2 cells 
We report that 7PA2 cells encapsulated in alginate microbeads can be effectively used for the sustained release of A&#946;1-42, and hence used to test the effect of chronic A&#946; secretion in any preclinical model. In Figure 5 below, we show the advantages of using alginate microbeads that can be easily injected and located within the brain of a rat. Ex vivo sections are here used for illustration purposes, comparing a millimeter alginate bead versus fabricated alginate microbeads.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59597/59597fig5.jpg" /> 
Figure 5: Using microbeads for relevant preclinical applications. Microbeads for engraftment in the rat brain must be small enough to be embedded without creating a lesion too large to avoid damage to the brain parenchyma and adversely affect normal brain function. Image (A) shows a size comparison between a millimeter-scaled bead and a micrometer-scaled bead side by side. (B) Implanting a millimeter-scaled bead within the brain for in vivo purposes would not work. Image (C) shows a microbead fabricated using this protocol. The size is suitable for insertion within the hippocampus of the rat without having a detrimental effect on normal physiology. <a href="https://www.jove.com/files/ftp_upload/59597/59597fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The images above highlight the importance of controlling the size of the microbead for such studies. Here, we demonstrate the advantage of using microbeads of diameters under 600 &#181;m. This allows the use of minimally invasive injection (e.g., Hamilton syringe) to better control the location of injected beads within the brain.
In summary, encapsulating 7PA2 cells gives control over the size of microbeads, number of encapsulated cells and the prediction of A&#946; secreted from the microbeads (e.g., concentration, release profile). Controlling the size of the microbeads is essential for two reasons: 1) to permit fine-tuned control over the concentration of released A&#946;, and 2) to allow implantation in a controlled region of the rat brain. The results obtained here describe the facile tuning of alginate microbeads and highlight potential applications for further studies.

Watch this Scientific Journal Video about Fabrication of Amyloid-ÃŽÂ²-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease at JoVE.com

Fabrication of Amyloid-&#946;-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-&#946; over time and can be used to study the effects of secreted amyloid-&#946; in in vitro and in vivo models.

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic ...

fabrication-amyloid-secreting-alginate-microbeads-for-use-modelling

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9.2K Views.  University of Manchester. This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-β over time and can be used to study the effects of secreted amyloid-β in in vitro and in vivo models.

Video: Fabrication of Amyloid-β-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

The use of Biofeedback in Clinical Virtual Reality: The INTREPID Project

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life activities. Its high prevalence in general population and the severe limitations it causes, point out the necessity to find new efficient strategies to treat it. Together with the cognitive-behavioral treatments, relaxation represents a useful approach for the treatment of GAD, but it has the limitation that it is hard to be learned. The INTREPID project is aimed to implement a new instrument to treat anxiety-related disorders and to test its clinical efficacy in reducing anxiety-related symptoms. The innovation of this approach is the combination of virtual reality and biofeedback, so that the first one is directly modified by the output of the second one. In this way, the patient is made aware of his or her reactions through the modification of some features of the VR environment in real time. Using mental exercises the patient learns to control these physiological parameters and using the feedback provided by the virtual environment is able to gauge his or her success. The supplemental use of portable devices, such as PDA or smart-phones, allows the patient to perform at home, individually and autonomously, the same exercises experienced in therapist's office. The goal is to anchor the learned protocol in a real life context, so enhancing the patients' ability to deal with their symptoms. The expected result is a better and faster learning of relaxation techniques, and thus an increased effectiveness of the treatment if compared with traditional clinical protocols.

The Project on Virtual Reality Intelligent Multi-sensor System for the Treatment of Anxiety-related Disorders (INTREPID) is aimed at developing a multi-sensor context-aware virtual reality system for the treatment of anxiety-related disorders.

Generalized anxiety disorder (GAD) is a psychiatric disorder characterized by a constant and unspecific anxiety that interferes with daily-life ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...