This study was supported by the US Department of Veterans Affairs (Washington D.C.), grants 0852048, 0731201, and 0854430 from the National Science Foundation (Arlington, VA), , and grant RO1 DK080897 from the National Institutes of Health (Bethesda, MD). Mr. Khanna received support from a generous donation by Mr. Edward Ross and Dr. Monica Moya from the Bill &amp; Melinda Gates Foundation (Seattle, WA).

The protocol here describes a three-step procedure for generating multilayered alginate microbeads (Figure 1). First, alginate microbeads are formed (Figure 2A). This procedure is described in section 1 below. Cells or proteins can be added to the microbeads in this step in order to act as a delivery system. The next step involves the formation of a permselective layer on the microbeads and is described in section 2. The final step involves the formation of an additional alginate layer and is described in section 3. This layer forms on the outside of the surface of the beads (Figure 2B) can be used to encapsulate and deliver therapeutic molecules (Figure 2C) to direct cellular response to the system following transplantation.
1. Alginate Microbead Preparation
<ol>
 <li>Prepare a 1.5% (w/v) LVM alginate solution by dissolving 15 grams of LVM alginate in 1 mL of inner layer alginate solution (25 mM HEPES buffer, 118 mM NaCl, 5.6 mM KCl, and 2.5 mM MgCl2 in DI water, adjusted to pH 7.4). Mix on a Vortexer until the alginate power has fully dissolved to form a clear, viscous solution. Note: This protocol describes conditions optimal for islet encapsulation within alginate microcapsules. 1 The concentration and composition of the alginate microspheres can be altered to tune properties for other applications (e.g. drug delivery, tissue engineering, etc.) Note: For islet encapsulation the islets can be added to the alginate solution at this step prior to loading in the microencapsulator. </li>
 <li>Prepare the crosslinking solution (22 mM CaCl2 in DI water) by dissolving 100 mM CaCl2 and 10 mM HEPES buffer in DI water and adjusting to pH 7.4. Note: other divalent cations such as Br2+, Sr2+ etc can be used instead of Ca2+, depending on the nature of alginate gelation desired.</li>
 <li>Set up the two-channel air jacket alginate microencapsulator by adding a 25-gauge needle to a syringe, and adjust the valves on each side to make sure the needle is in the center of the air jacket. Different gauge needles can be used for this step, depending on the size of alginate microbeads targeted. 
 This step can also be performed with a syringe, should a microencapsulator not be available. Add the alginate solution to the syringe, and select a needle gauge based on the size of microbeads desired. </li>
 <li>Place a flask containing 10 mL of crosslinking solution directly underneath the needle. Place a stir bar in the solution. </li>
 <li>Inject droplets directly into the CaCl2 solution in order to crosslink the alginate to form microbeads. Incubate the beads in the crosslinking solution to cure for at least fifteen minutes, with continuous stirring. </li>
 <li>Transfer the beads to a 15 mL centrifuge tube. Remove the residual solution, and perform three washes with a 0.2% CaCl2 in normal saline solution for two minutes each. </li>
</ol>
2. Coating the Microbeads with Poly-L-ornithine
<ol>
 <li>Prepare 3 mL of a 0.1% (w/v) solution of poly-L-ornithine (PLO) in normal saline. Place the solution on a vortex until it is completely dissolved, forming a clear solution. Poly-L-lysine (PLL) can be used instead of PLO for this step, which results in similar levels of permselectivity. </li>
 <li>Transfer the alginate microbeads into the PLO solution. Place on a Vortex for 30 minutes to allow the PLO sufficient time to interact with the alginate, forming a polycation-polyanion complex. At the end of 30 minutes, there should be a distinctly visible white coating around the alginate microbeads. </li>
 <li>Remove the PLO solution, and perform three washes with 0.2% CaCl2 in normal saline for two minutes each. </li>
</ol>
3. Creating the Outer Alginate Layer
<ol>
 <li>Prepare an alginate solution of desired concentration that is to be used to create the outer layer. The solution is prepared as described in (1.1) above. The size of the outer layer is influenced by the composition and concentration of alginate used (Figure 3). </li>
 <li>Transfer the alginate microbeads to a cell-strainer. Use a Kimwipe to absorb excess solution in order to dry the microbeads as much as possible. </li>
 <li>Transfer the microbeads to a Parafilm surface. Other smooth surfaces such as glass or even a plastic Petri dish can be substituted for Parafilm, if desired. </li>
 <li>Transfer the alginate solution prepared in (3.1) onto the alginate microbeads. The volume of alginate solution be should completely cover the alginate microbeads. Let the microbeads remain in the alginate solution for 45 minutes. </li>
 <li>Use a pipette to remove the excess alginate solution not bound to the microbeads. </li>
 <li>Transfer the microbeads into a solution of 22mM CaCl2. This crosslinks the alginate solution around the microbeads, resulting in the formation of the distinct outer alginate layer. </li>
 <li>Perform three washes with 22 mM CaCl2 in normal saline for two minutes each. Viewing the microbeads under a microscope should allow for a distinct outer alginate layer to be seen formed around the core microbead. </li>
</ol>
4. Representative Results
<img src="/files/ftp_upload/3388/3388fig1.jpg" alt="Figure 1" /> 
Figure 1. A schematic of the procedure for creating multilayered alginate microbeads. Reproduced with permission from Khanna et al. J Biomed Mater Res A. Nov; 95(2), 632-40 (2010).
 <img src="/files/ftp_upload/3388/3388fig2.jpg" alt="Figure 2" /> 
Figure 2. (A) and (B) are phase contrast images of alginate microbeads. (A) shows a microbead after synthesis step (1.7), while (B) displays a distinct outer alginate layer present after completion of step (3.7). (C) is a FITC image of fluorescently-labeled BSA protein encapsulated in the outer layer. Reproduced with permission from Khanna et al. J Biomed Mater Res A. Nov;95(2), 632-40 (2010).
<img src="/files/ftp_upload/3388/3388fig3.jpg" alt="Figure 3" /> 
Figure 3. The size of the outer alginate layer can be varied based on the composition and concentration of alginate used. Our results show that, for both LVM and LVG alginate, the outer layer size increases with increasing alginate concentration, and that LVG alginate yields thicker outer layers than LVM alginate at equal concentrations. Reproduced with permission from Khanna et al. J Biomed Mater Res A. Nov; 95(2), 632-40 (2010).
 <img src="/files/ftp_upload/3388/3388fig4.jpg" alt="Figure 4" /> 
 Figure 4. Release of FGF-1, an angiogenic growth factor protein, from the outer alginate layer varied based on the concentration of LVM and LVG alginate used. (A) and (B) show percent release, and (C) and (D) denote the corresponding mass release of FGF-1 versus time for different outer layer formulations. There is a burst release exhibited for all conditions within the initial 5 h (A and C), and low-dose continuous release for up to 30 days (B and D). Reproduced with permission from Khanna et al. J Biomed Mater Res A. Nov; 95(2), 632-40 (2010). <a href="https://www.jove.com/files/ftp_upload/3388/3388fig4large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Darrabie, M. D., Kendall, W. F., Opara, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+Poly-L-+Ornithine-coated+alginate+microcapsules.">Characteristics of Poly-L- Ornithine-coated alginate microcapsules.</a> Biomaterials. 26, 6846-6852 (2005).</li><li>Yamagiwa, K., Kozawa, T., Ohkawa, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+alginate+composition+and+gelling+conditions+on+diffusional+and+mechanical+properties+of+calcium-alginate+gel+beads.">Effects of alginate composition and gelling conditions on diffusional and mechanical properties of calcium-alginate gel beads.</a> J. Chem. Eng. Jpn. 28, 462-467 (1995).</li><li>Amsden, B., Turner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+characteristics+of+calcium+alginate+gels.">Diffusion characteristics of calcium alginate gels.</a> Biotechnol. Bioeng. 65, 605-610 (1999).</li><li>Uriel, S., Brey, E. M., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+low+levels+of+fibroblast+growth+factor-1+promote+persistent+microvascular+network+formation.">Sustained low levels of fibroblast growth factor-1 promote persistent microvascular network formation.</a> Am. J. Surg. 192, 604-609 (2006).</li><li>Moya, M. L., Lucas, S., Francis-Sedlak, M., Liu, X., Garfinkel, M. R., Huang, J. J., Cheng, M. H., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+delivery+of+FGF-1+increases+vascular+density+in+comparison+to+bolus+administration.">Sustained delivery of FGF-1 increases vascular density in comparison to bolus administration.</a> Microvasc. Res. 78, 142-147 (2009).</li><li>Moya, M. L., Cheng, M. H., Huang, J. J., Francis-Sedlak, M. E., Kao, S. W., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+FGF-1+loaded+alginate+microbeads+on+neovascularization+and+adipogenesis+in+a+vascular+pedicle+model+of+adipose+tissue+engineering.">The effect of FGF-1 loaded alginate microbeads on neovascularization and adipogenesis in a vascular pedicle model of adipose tissue engineering.</a> Biomaterials. 31, 2816-2826 (2010).</li><li>Khanna, O., Moya, M. L., Greisler, H. P., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayered+Microcapsules+for+the+Sustained-Release+of+Angiogenic+Proteins+From+Encapsulated+Cells.">Multilayered Microcapsules for the Sustained-Release of Angiogenic Proteins From Encapsulated Cells.</a> Am. J. Surg. 200, 655-658 (2010).</li><li>Khanna, O., Moya, M. L., Opara, E. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+multilayered+alginate+microcapsules+for+the+sustained-release+of+fibroblast+growth+factor-1.">Synthesis of multilayered alginate microcapsules for the sustained-release of fibroblast growth factor-1.</a> J. Biomed. Mater. Res. A. 95, 632-640 (2010).</li></ol>

No conflicts of interest declared.

Alginate is a natural, acidic polysaccharide extracted from algae and is composed of units of 1,4'-&beta;-D-mannuronic acid (M) and &#x03B1;-L-guluronic acid (G) 2,3. Simple gelation occurs when divalent cations, such as Ca2+, Sr2+, or Ba2+ interact with G-monomers forming ionic bridges between adjacent alginate chains. Microbeads of alginate have been utilized to deliver a variety of proteins, including fibroblast growth factor-1 (FGF-1), nerve growth factor, leukemia inhibiti...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Pronova Ultrapure LVG alginate</td>
 <td>Nova-Matrix</td>
 <td>4200006</td>
 <td>A variety of alginate 			formulations are available. The choice of alginate influences the 			end properties of the microbeads, including size, mechanical 			properties, and transport. The composition used should be 			optimized for a given application. </td>
 </tr>
 <tr>
 <td>Pronova Ultrapure LVM alginate</td>
 <td>Nova-Matrix</td>
 <td>4200206</td>
 <td>A variety of alginate 			formulations are available. The choice of alginate influences the 			end properties of the microbeads, including size, mechanical 			properties, and transport. The composition used should be 			optimized for a given application. </td>
 </tr>
 <tr>
 <td>Poly-L-ornithine hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>P2533</td>
 <td></td>
 </tr>
 </tbody>
</table>

generation of alginate microspheres for biomedical applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

Watch this Scientific Journal Video about Generation of Alginate Microspheres for Biomedical Applications at JoVE.com

Generation of Alginate Microspheres for Biomedical Applications

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

generation-of-alginate-microspheres-for-biomedical-applications

Research

JoVE Journal

Medicine

20.6K Views.  Illinois Institute of Technology. In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Video: Generation of Alginate Microspheres for Biomedical Applications

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...