Name: procedure to evaluate the efficiency of flocculants for the removal of dispersed particles from plant extracts
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I would like to acknowledge Dr. Thomas Rademacher for providing the transgenic tobacco seeds and Ibrahim Al Amedi for cultivating the tobacco plants. I wish to thank Dr. Richard M Twyman for editorial assistance and Prof. Dr. Rainer Fischer for fruitful discussions. This work was funded in part by the European Research Council Advanced Grant ''Future-Pharma'', proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and the Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.

1. Develop an Adequate Experimental Strategy
<ol>
	<li>Identify the environmental and process parameters that are relevant for the flocculation procedure to be established or optimized, i.e. which factors have the strongest effect on flocculation. Typically, there are several such parameters so a DoE approach as recently described 20 is necessary due to the lack of mechanistic models.
	<ol>
		<li>Select parameters (factors) based on literature data 12, prior knowledge and experience with ...

<ol>
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The most important aspect to consider when setting up a DoE to characterize particle flocculation is that the design must in principle be able to detect and describe the anticipated or possible effects 36,38, e.g. the influence of pH, polymer type and polymer concentration 16. Therefore, it is important to evaluate the fraction of design space (FDS) before starting the actual experiments. The FDS is the fraction of the multidimensional experimental space (covered by the design factors, ...

Plants are widely used to produce food commodities such as fruit juices, but they can also be developed as platforms for the manufacture of higher-value biopharmaceutical products 1-3. In both cases, downstream processing (DSP) often begins with the extraction of liquids from tissues such as leaves or fruits, followed by the clarification of particle-laden extracts 4,5. For the manufacture of biopharmaceuticals, the costs of DSP can account for up to 80% of the overall production costs 6,7 and this in part reflects the high particle burden present in extracts prepared by disruptive methods such as blade-based homogenization 8,9. Although the rational selection of filter layers to match the particle size distribution in the extract can increase filter capacity and reduce costs 10,11, the improvement can never exceed the ceiling of absolute capacity defined by the number of particles that must be retained per unit of filter area to achieve clarification.
The ceiling can be lifted if fewer particles reach the surface of the finest filters in the filtration train, and this can be achieved if dispersed particles are mixed with polymers known as flocculants that promote aggregation to form large flocs 12. Such flocs can be retained further upstream by coarser and less expensive bag filters, reducing the particle burden reaching the finer and more expensive depth filters. The polymers must have safety profiles suitable for their applications, e.g. for biopharmaceuticals they must be compliant with good manufacturing practice (GMP), and typically they must have a molar mass &#62;100 kDa and can either be neutral or charged 13. Whereas neutral flocculants generally act by cross-linking dispersed particles causing their aggregation and the formation of flocs with diameters &#62;1 mm 11, charged polymers neutralize the charge of dispersed particles, reducing their solubility and thus causing precipitation 14.
Flocculation can be improved by adjusting parameters such as buffer pH or conductivity, and the polymer type or concentration, to match the properties of the extract 15,16. For tobacco extracts pretreated with 0.5-5.0 g L-1 polyethylenimine (PEI), a greater than 2-fold increase in depth filter capacity was reported in a 100-L pilot-scale process. The cost of this polymer is less than &#8364;10 kg-1 so its introduction into the process resulted in cost savings of about &#8364;6,000 for filters and consumables per batch 16 or even more when combined with cellulose-based filter aids 17. Even so, predictive models are required to evaluate the a priori economic benefits of flocculants because their inclusion can require hold steps of 15-30 min 16,18, resulting in further investment costs for storage tanks. However, there are currently no mechanistic models available that can predict the outcome of such experiments due to the complex nature of flocculation. Therefore, a more appropriate design-of-experiments (DoE) approach 19 was developed as described in this article. A protocol for the general DoE procedure has recently been published 20.
Small-scale devices are now available for the high-throughput screening of flocculation conditions 21. However, these devices may not realistically simulate conditions during the flocculation of plant extracts because the dimensions of the reaction vessel (~7 mm for wells on a 96-well plate) and the particles or flocs can be less than an order of magnitude apart. This can affect mixing patterns and thus the predictive power of the model. Furthermore, it can be difficult to scale down processes involving precipitation due to non-linear changes in the mixing behavior and precipitate stability 22. Therefore, this article outlines a bench-top-scale screening system with a throughput of 50-75 samples per day, yielding results that are scalable from the initial 20 ml reaction volume to a 100 L pilot-scale process 16. When combined with a DoE approach, this allows the predictive models to be used for process optimization and documentation as part of a quality-by-design concept.
The method described below may also be adapted to biopharmaceuticals produced in cell culture-based processes, where flocculants are also being considered as a cost-saving tool 23. It can also be used to model the precipitation of target proteins from a crude extract as part of a purification strategy, as demonstrated for &#946;-glucuronidase produced in canola, maize and soybean 24,25. A detailed description of flocculant properties can be found elsewhere 16,26 and it is important to ensure that the polymer concentrations are either non-toxic or below harmful levels in the final product 11.

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			<td height="21" style="height:21px;width:360px;">Biacore T200</td>
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			<td style="width:119px;">28-9750-01</td>
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			<td height="21" style="height:21px;width:360px;">BP-410</td>
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			<td height="21" style="height:21px;width:360px;">Catiofast VSH</td>
			<td style="width:195px;">BASF</td>
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			<td height="21" style="height:21px;width:360px;">Centrifuge 5415D</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">5424 000.410</td>
			<td>Centrifuge</td>
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			<td height="21" style="height:21px;width:360px;">Centrifuge tube 15 mL</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">2017106</td>
			<td>Reaction tube</td>
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			<td height="21" style="height:21px;width:360px;">Centrifuge tube 50 mL self-standing</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">1110504</td>
			<td>Reaction tube</td>
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			<td height="21" style="height:21px;width:360px;">Chitosan</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">5375.1</td>
			<td>Flocculating agent</td>
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			<td height="21" style="height:21px;width:360px;">Design-Expert(R) 8</td>
			<td style="width:195px;">Stat-Ease, Inc.</td>
			<td style="width:119px;">n.a.</td>
			<td>DoE software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium phosphate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">&#160;4984.3&#160;</td>
			<td>Media component</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
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			<td style="width:119px;">5.220072</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Forma -86C ULT freezer</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">88400</td>
			<td>Freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Greenhouse</td>
			<td style="width:195px;">n.a.</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Grodan Rockwool Cubes 10x10cm</td>
			<td style="width:195px;">Grodan</td>
			<td style="width:119px;">102446</td>
			<td>Rockwool block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HEPES</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9105.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">K700P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">5302305</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">KS50P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">B12486</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Miracloth</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">475855-1R</td>
			<td>Filter cloth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">MultiLine Multi 3410 IDS</td>
			<td style="width:195px;">WTW</td>
			<td style="width:119px;">WTW_2020</td>
			<td>pH meter / conductivity meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Osram cool white 36 W</td>
			<td style="width:195px;">Osram</td>
			<td style="width:119px;">4930440</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phytotron</td>
			<td style="width:195px;">Ilka Zell</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Polymin P</td>
			<td style="width:195px;">BASF</td>
			<td style="width:119px;">79002360</td>
			<td>Flocculating agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">POLYTRON PT 6100 D</td>
			<td style="width:195px;">Kinematica</td>
			<td style="width:119px;">11010110</td>
			<td>Homogenization device with custom blade tool</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Protein A</td>
			<td style="width:195px;">Life technologies</td>
			<td style="width:119px;">10-1006</td>
			<td>Antibody binding protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium chloride</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">P029.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Synergy HT</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;">SIAFRT</td>
			<td>Fluorescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TRIS</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4855.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tween-20</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9127.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">VelaPad 60</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">VP60G03KNH4</td>
			<td>Filter housing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Nano ZS</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">ZEN3600</td>
			<td>DLS particle size distribution measurement</td>
		</tr>
	</tbody>
</table>

procedure to evaluate the efficiency of flocculants for the removal of dispersed particles from plant extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

Flocculation of tobacco extract with different polymers
The method described above was successfully used to develop a process for the flocculation of tobacco extracts during the manufacture of a monoclonal antibody (the HIV-neutralizing antibody 2G12) and a fluorescent protein (DsRed) (Figure 1) 16, and has since been transferred to other proteins including lectins, malaria vaccine candidates and fusi...

Watch this Scientific Journal Video about Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts at JoVE.com

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

The overall goal of the following experiment is to identify flocculants that can improve the removal of dispersed particles from plant extracts. This is achieved by setting up an experimental design to identify the most effective polymers and parameters controlling the aggregation of these dispersed particles. The second step defines the work flow, which corresponds to the unit operations used in a large-scale application.Next, leaf extracts are prepared for flocculants under various conditions and analyzed in order to measure turbidity and product recovery. The results show the optimal flocculation conditions and polymers based on a design of experiments model evaluation, which we shorten to DoE. The main advantage of this technique compared to traditional clarification using depth-filtration alone, is that the filter capacity can be increased several-fold, reducing overall process costs.Flocculation can increase the effective diameter of particles and facilitate their removal by centrifugation or filtration. Therefore, the environmental process parameters that are relevant for the flocculation procedure must be identified first. Typically, there are several such parameters.So a DoE approach is appropriate given the lack of mechanistic models. The DoE is set up by selecting parameters based on literature data and prior knowledge. Typical parameters include incubation time as well as polymer type and concentration.The experimental outcomes that are used to evaluate the efficiency of flocculation, such as filtratability, are also defined before the experiment. This information is then fed into a DoE type appropriate for the number of factors to be investigated and the degree of knowledge already accumulated about the system. A work flow is developed to encompass all steps relevant for the flocculation and laboratory-scale processing of plant extracts.The aliquot volume should be small enough to allow high sample throughput. The performance of the laboratory equipment should correlate with that of the process-scaled devices. Plants are cultivated in a manner that represents the final process.And all utilities are prepared before starting the extraction and flocculation experiments. The flocculant stock solutions are prepared carefully taking into account any pre-dilution by the manufacturer. Choosing the same stock concentration for all flocculants simplifies subsequent pipetting, reducing the likelihood of errors.The buffer components, pH, and conductivity for each fresh flocculant stock solution are prepared to match the conditions of the extracts. The DoE schedule is converted into a pipetting scheme setting out the volumes of stock solutions to be added to the plant extract samples. Plant extract samples are prepared from harvested tobacco leaves using the buffers matching the conditions to be tested in the DoE.While stirring the extract, aliquots are prepared, matching the pre-defined volume. This will ensure homogeneous particle distribution in all samples. Extraction buffer is added to each aliquot so that every sample has the same volume once a flocculant stock has been added.The volume of stock solution defined in the DoE is added to the sample sequentially as indicated by the randomized run order. The polymer is homogeneously distributed throughout the sample by shaking the tube immediately after adding the polymer, which will also ensure that the flocculation results are reproducible. The method can easily be modified to test combinations of flocculants or the repeated addition of the same flocculant.But this must be implemented in the DoE. Floc formation typically requires three to 30 minutes incubation time. Sticking to the incubation times defined in the DoE is necessary to achieve reproducible results.Either the settling velocity of the flocs or the turbidity of the filtrates and filter capacities can be used to evaluate the flocculant performance. Confirming that the polymers do not interfere with products such as proteins, is important too. Once the data quality is confirmed, information can be transferred into the DoE software.The experimental data are analyzed using the tools built into the DoE software including data transformation, factor selection, and quality control tools. As well as using these tools, the final model should also agree with the basic laws of physics and thermodynamics. Once the model quality is confirmed, the model can be used to predict the optimal conditions for flocculation.Based on the model predictions, the design space can be narrowed down to the most relevant region for which a new DoE can be set up, repeating the procedure described before. The results can then be confirmed at the pilot or production scale. In this representative study, the performance of 23 different synthetic and four natural flocculants was tested for their ability to flocculate tobacco leaf extracts using a DoE approach.Taking into account polymer-specific parameters such as polymer concentration and type, but also process parameters, such as incubation time, temperature, buffer pH, and conductivity. With the sequence of four nested IV-optimal designs, including a total of about 200 20 mL extract aliquots, we found that Polymin P was the most effective flocculant for tobacco extracts in the pH range four to eight, as long as the temperature was between four and 30 degrees Celsius. An incubation time of 15 minutes was sufficient for flocculation, but the effectiveness declined at conductivities greater than 40 mS per cm.However, the DoE approach showed that Catiofast VSH was complementary to Polymin P, achieving effective flocculation at conductivities greater than 40 mS per cm. When these flocculants were introduced into the downstream processing of tobacco extracts, the capacity of the deployed depth filters increased 3.2 to 5.7-fold, reducing the cost of goods for this step accordingly. We confirmed that the flocculants did not reduce the recovery of target proteins including a monoclonal antibody, a fluorescence protein, a lactin, and different vaccine candidates.The optimal flocculation conditions were also confirmed in a 100 L pilot scale batch. The results of the DoE were also successfully transferred to tobacco extracts prepared with a screw press rather than a blade-based homogenizer underlining how broadly applicable the collected information can be. We also adapted the method to test combinations of flocculants which worked equally well.After watching this video, you should understand how to set up flocculation experiments for plant extracts using a DoE approach.

procedure-to-evaluate-efficiency-flocculants-for-removal-dispersed

Research

JoVE Journal

Biology

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Assessing the Putative Anticryptococcal Properties of Crude and Clarified Extracts from Mollusks

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The widespread use of antifungals in clinical settings, their use in agriculture, and strain hybridization have led to increased evolution of resistance. This rising rate of resistance against antifungals is a growing concern among clinicians and scientists worldwide, and there is heightened urgency to develop novel antifungal therapies. For instance, C. neoformans produces several virulence factors, including intra- and extra-cellular enzymes (e.g., peptidases) with roles in tissue degradation, cellular regulation, and nutrient acquisition. The disruption of such peptidase activity by inhibitors perturbs fungal growth and proliferation, suggesting this may be an important strategy for combating the pathogen. Importantly, invertebrates such as mollusks produce peptidase inhibitors with biomedical applications and anti-microbial activity, but they are underexplored in terms of their usage against fungal pathogens. In this protocol, a global extraction from mollusks was performed to isolate potential peptidase inhibitors in crude and clarified extracts, and their effects against classical cryptococcal virulence factors were assessed. This method supports the prioritization of mollusks with antifungal properties and provides opportunities for the discovery of anti-virulence agents by harnessing the natural inhibitors found in mollusks.

The human fungal pathogen Cryptococcus neoformans produces a variety of virulence factors (e.g., peptidases) to promote its survival within the host. Environmental niches represent a promising source of novel natural peptidase inhibitors. This protocol outlines the preparation of extracts from mollusks and the assessment of their effect on fungal virulence factor production.

Cryptococcus neoformans is an encapsulated human fungal pathogen with a global distribution that primarily infects immunocompromised individuals. The ...

Quantitative Analysis of Microbial Autoaggregation Using Imaging Flow Cytometry

Imaging Flow Cytometry to Study Microbial Autoaggregation

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The Lactobacillaceae family consists of Gram-positive bacteria with confirmed probiotic properties. This study utilizes Lactobacillaceae species as a model to demonstrate the effectiveness of single-cell high throughput analysis in studying cellular aggregation. The focus is on analyzing the response of these beneficial species to simple carbohydrates from the diet.
The study showcases how Imaging Flow Cytometry (IFC) can overcome the fundamental differences in the assembly of probiotic bacteria in the presence and absence of carbohydrates. IFC combines the power and speed of conventional flow cytometry with the spatial resolution of microscopy, enabling high-rate complex morphometric measurements in a phenotypically defined manner across a library of beneficial bacterial strains and conditions. This protocol provides insights into the autoaggregation of Lactobacillaceae species and sheds light on their response to dietary carbohydrates, contributing to understanding the mechanisms behind the beneficial effects of these probiotic bacteria.

Author Spotlight: Advancing Research in Microbial Autoaggregation Using Imaging Flow Cytometry

This protocol describes a quantitative approach to measure microbial autoaggregation using imaging flow cytometry.

Beneficial and probiotic bacteria play essential roles in their hosts, providing various health benefits, including immunity to infectious diseases. The ...

&lt;em&gt;Drosophila&lt;/em&gt; Fecal Deposit Analysis for Drug Discovery

Assessment of The Effect of Antidiarrheal Drugs and Plant Extracts on Drosophila melanogaster

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a model to study intestinal function, only a few reports have focused on Drosophila melanogaster (D. melanogaster). Compared to mice, fruit flies present many advantages, such as a short life cycle, cost-effective and simple maintenance, and no ethical issues. Furthermore, the mammalian gastrointestinal physiology, anatomy, and signaling pathways are highly conserved in D. melanogaster. Plant extracts have been used traditionally to treat diarrhea and constipation. For example, Psidium guajava (P. guajava) is one of the most known antidiarrheal agents in the tropics. However, no studies have evaluated the effect of antidiarrheal and laxative drugs and plant extracts in D. melanogaster, and it remains unknown if similar effects (e.g., smaller, more concentrated, and less abundant fecal deposits in the case of antidiarrheal drugs) can occur in the fruit flies compared to mammals. In this study, an antidiarrheal effect induced by P. guajava is demonstrated in a D. melanogaster strain that presents a diarrheic phenotype. Fecal sampling produced by flies is monitored using a dye-supplemented food. This protocol outlines the method used for preparing food with drugs, evaluating the fecal deposits of flies fed on these food preparations, and interpreting the data obtained.

Author Spotlight: Advancing Antidiarrheal Research with the Drosophila Model 

Here, a method is described to feed Drosophila melanogaster with drugs and plant extracts and assess their effect on the gastrointestinal tract by analyzing the fruit flies fecal deposits. The drug-treated flies can be used as a model for further research.

To study human gastrointestinal physiology, biomedical scientists have relied on the use of model organisms. Although many researchers have used mice as a ...

Dissection and Removal of the Temporal Bone to Collect Auditory Epithelia from Newborn Mice

To begin, securely hold the head of the euthanized newborn mice in place. Using micro operating scissors, open the scalp along the sagittal suture. Then, separate and immobilize the scalp bilaterally with the fingers.Use scissors to sever the cranium and flip it forward to access the skull. Employ a small periosteal elevator to extract the brain and then use the tip of the scissors to gently scrape the brain, revealing the base of the skull. Now, examine the bilateral temporal bones at the skull's base.Employ scissors to incise the base of the skull along the midline. Then, scrape off the skin and eliminate any unnecessary bone. Preserve and transfer the temporal bones to 35-millimeter sterile Petri dishes filled with fresh HBSS.Next, use a pair of 5 number pointed forceps to eliminate the bola and surrounding tissue from the petrous portion of the temporal bone. Hold the forceps with one hand to stabilize the semi-circular part of the temporal bone. Use the other hand to insert the lower tip of the forceps into the round window niche, separating the lateral bone of the cochlea from the scale of vestibule.Carefully remove the petrous portion of the temporal bone without contacting the Organ of Corti epithelium. Then, meticulously detach and extract the bony labyrinth of the cochlea, starting from the basal to the apical end. Using 5 number pointed forceps, carefully micro isolate the sensory epithelium of the Organ of Corti from the modiolus.Next, employ micro-operating forceps to hold the spiral ligament, and gently separate it from the stria vascularis. Transfer the clean auditory epithelium, using a 200 microliter pipette, to a three millimeter sterile culture dish containing HBSS. Place the isolated auditory epithelium in a 100 millimeter sterile Petri dish filled with HBSS for the subsequent preparation steps.