This work was supported by NIH/NICHD HD091068-01 to Dr. Ina Dobrinski.

NOTE: Testes from 1-week-old piglets were obtained from a commercial pig farm as by-product from castration of commercial pigs. Sourcing of testes was approved by the Animal Care Committee at the University of Calgary.
1. Preparation of enzyme solutions for tissue digestion
NOTE: Three different enzymatic solutions are needed, which include two different collagenase IV solutions (solution A, B) and a deoxyribonuclease I (DNase I) solution.
<ol>
	<li>To prepare solution A, dissolve 20 mg of collagenase IV S (Table of Materials) and 40 mg of collagenase IV W (Table of Materials) in 25 mL of high glucose Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM). Then filter sterilize with a 0.22 &#181;m filter. Add 0.4 mL of fetal bovine serum (FBS) to solution A to inhibit trypsin activity.</li>
	<li>To prepare solution B, dissolve 80 mg of collagenase IV W (Table of Materials) in 40 mL of DMEM. Then filter sterilize with a 0.22 &#181;m filter.</li>
	<li>To prepare DNase I solution (7 mg/mL), dissolve 70 mg of DNase I in 10 mL of DMEM. Then filter sterilize with a 0.22 &#181;m filter. 
	NOTE: The enzyme concentration for collagenase IV solutions outlined here, varies from the concentrations (2 mg/mL) stated in the original article14. The current protocol yields cells with slightly higher viability. However, cells isolated by both protocols produce organoids with identical tissue architecture.</li>
</ol>
2. Testis tissue enzymatic digestion
<ol>
	<li>Collect the testes into a sterile beaker and wash with phosphate buffered saline (PBS) containing 1% penicillin/streptomycin (P/S). After washing, transfer the testes to a 100 mm tissue culture dish with PBS containing 1% P/S and remove the tunica vaginalis and epididymis using autoclaved scissors and forceps. Transfer the isolated testes to a new 100 mm dish and wash thoroughly with PBS containing 1% P/S.</li>
	<li>To maintain sterility, use another set of sterile scissors and forceps to peel the testicular parenchyma out of the tunica albuginea. Cut the testes along the longitudinal axis directly under the tunica. Then peel the testes out of the tunica using two forceps and place into a new 100 mm dish containing of 1 mL DMEM with 1% P/S.</li>
	<li>Mince the peeled testes with sterile scissors into 1-2 mm tissue pieces. After chopping, use sterile forceps to remove white fragments of connective tissue.</li>
	<li>Transfer the minced tissue pieces into solution A and top it up to 50 mL with DMEM to obtain a concentration of 0.4 mg/mL for collagenase IV S (Table of Materials) and a concentration of 0.8 mg/mL for collagenase IV W (Table of Materials). Place solution A containing the tissue pieces into a 37 &#176;C water bath for 30 min, gently invert the tubes every 5 min and check visually for the release of DNA.
	<ol>
		<li>Add 500 &#181;L of DNase I if free floating DNA is observed. After 30 min, centrifuge the tube at 90 x g with brakes at 25 &#176;C for 1.5 min and discard the supernatant. 
		NOTE: DNA would appear as a cloudy substance. When the tubes are shaken, the tissue pieces settle down whereas the DNA would stay afloat.</li>
	</ol>
	</li>
	<li>Add solution B to the tube and top up to 50 mL with DMEM to obtain a concentration of 1.2 mg/mL of collagenase IV W (Table of Materials). Place the tube into 37 &#176;C water bath for 30 min and gently invert the tube every 5 min. Add 500 &#181;L of DNase I if free floating DNA is observed.</li>
	<li>After 30 min, centrifuge the tube at 90 x g with brakes at 25 &#176;C for 1.5 min. After that discard the supernatant and wash once with PBS with 1% P/S. 
	NOTE: The discarded supernatants from both solution A and B will primarily contain interstitial cells.</li>
	<li>Top up the tube with PBS up to 50 mL. Carefully collect the tubules from the top and place into a new 50 mL tube. 
	NOTE: The big undigested tissue fragments quickly settle at the bottom, while digested seminiferous tubules will remain in suspension. No big tissue fragments should be collected. This procedure can be repeated several times until the solution in the original tube is almost clear and just undigested tissue fragments are remaining, which can be disposed.</li>
	<li>Centrifuge the tubules at 90 x g with brakes at 25 &#176;C for 1.5 min and discard the supernatant. Add fresh PBS and centrifuge again. Repeat this wash step twice.</li>
	<li>After the last PBS wash, remove the supernatant and resuspend the seminiferous tubules in 5 mL of PBS. Then add 15 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) to the tubules. Place the tube in 37 &#176;C water bath and gently invert every 2 min. If a lot of free-floating DNA is observed, add 500 &#181;L of DNase I with 5 mL of DMEM.</li>
	<li>Evaluate the enzymatic digestion of tubules to single cells under the microscope (first after 5 min, then every 2 min). If mostly single cells can be detected, stop the reaction with 5 mL of FBS and filter through a 70 &#181;m mesh and then through a 40 &#181;m mesh. 
	NOTE: Care should be taken that the caps on the 50 mL tubes used for digestions (Solution A, B and 0.25% trypsin-EDTA) are tightened properly. If contamination in the water bath is a concern, paraffin film may be wrapped around the cap to ensure sterility.</li>
	<li>Centrifuge the single cells at 500 x g with brakes at 25 &#176;C for 5 min, resuspend in the enrichment medium (Dulbecco Modified Eagle Medium F/12 (DMEM/F12) containing 5% FBS and 1% P/S) and count the number of viable cells. This is the starting cell population. Fix 100 x 103 cells and perform immunocytochemistry for germ cell marker UCHL1 (Ubiquitin C-Terminal Hydrolase L1) as described12 and determine the percentage of germ cells in this starting cell population. 
	NOTE: Germ cell marker Promyelocytic leukemia zinc finger protein (PLZF)15 could also be used as a suitable alternative for UCHL1. In the starting cell population, the expected cell yield is around 700-800 x 109/g of tissue. The percentage of germ cells in this cell population should be 4-5%.</li>
	<li>Place around 20 x 106 of this starting cell population in two ultra low attachment 100 mm Petri dishes (10 x 106 cells suspended in 10 mL of DMEM/F12 containing 1% P/S in each dish) for 2 days in a tissue culture incubator (37 &#176;C, 5% CO2, 21% oxygen). Perform the germ cell enrichment with the remaining cells. 
	NOTE: To ensure optimum viability and cell quality, while the germ cell enrichment and subsequent quantification takes place, the starting cell population is placed in culture in ultra low attachment tissue culture dishes for 2 days.</li>
</ol>
3. Germ cell enrichment
NOTE: The procedure described above yields primarily Sertoli cells and germ cells. Different adhesion properties allow for the separation of Sertoli cells and germ cells via differential plating.
<ol>
	<li>Seed 20-25 x 106 cells of the starting cell population per 100 mm tissue culture dish in a total volume of 8 mL of enrichment medium (DMEM/F12 with 5% FBS and 1% P/S) for differential plating. Place the cells into an incubator (37 &#176;C, 5% CO2, 21% O2) and after 1.5 h, ensure that the majority of Sertoli cells attach to the plate.</li>
	<li>Collect and combine the supernatant (mainly containing germ cells) of 2 plates to one new 100 mm plate and place it back into the incubator. After 1 h, again combine the supernatant of 2 plates to a new 100 mm plate. Place the plates back into the incubator for overnight. 
	NOTE: The supernatants combined will primarily contain germ cells. The adhered cells that are discarded along with the plates are primarily testicular somatic cells such as Sertoli and peritubular myoid cells.</li>
	<li>Collect the enriched germ cells in two fractions as follows. 
	NOTE: Enriched germ cells adhere differently, non adherent fraction and slightly adhered fraction. Both these fractions together form the enriched germ cell population
	<ol>
		<li>Non adherent fraction: collect the supernatant.</li>
		<li>Slightly adherent fraction: wash the plates gently with PBS, treat with 2 mL of 1:20 dilution of 0.25% trypsin-EDTA for 5 min at room temperature, stop the reaction with 2 mL of enrichment medium and collect in the same tube as non adherent fraction. 
		NOTE: 0.25% Trypsin-EDTA needs to be diluted with PBS to produce a 1:20 trypsin solution.</li>
	</ol>
	</li>
	<li>Count the total number of cells using a cell counter, fix 100 x 103 cells and perform immunocytochemistry for germ cell marker UCHL1 as described12 and determine the percentage of germ cells in this enriched cell population. 
	NOTE: The percentage of germ cells in this cell population should be at least 60-70%. Since the immunocytochemistry for quantification would require 1 day, the enriched cells should be placed in an ultra low attachment 100 mm Petri dish with DMEM/F12 supplemented with 1% P/S for the duration to ensure optimum cell quality and viability.</li>
</ol>
4. Preparation of cells for seeding
<ol>
	<li>To harvest the starting cell preparation, collect the supernatant in a 50 mL tube from the ultra low attachment 100 mm dishes. Wash the plates vigorously with PBS to collect the adhered cells. No enzymatic digestion is needed. 
	NOTE: Some of the testicular cells, primarily Sertoli cells, can slightly adhere to the ultra low attachment plates.</li>
	<li>Combine the starting cell preparation and enriched germ cells to obtain a working cell preparation containing 25% germ cells. Centrifuge at 500 x g with brakes at 25 &#176;C for 5 min, discard the supernatant and resuspend the cells in organoid formation medium (DMEM/F12 supplemented with insulin 10 &#181;g/mL, transferrin 5.5 &#181;g/mL, selenium 6.7 ng/mL, 20 ng/mL epidermal growth factor, 1% P/S). Adjust the cell density to 2.4 x 106 per mL. 
	NOTE: Each well in the microwell plates used13 in this protocol contained 1,200 microwells.</li>
	<li>Use the formula below to calculate the number of cells per well as described below. 
	Number of cells per well = number of microwells x number of cells to be clustered for each organoid.</li>
	<li>To generate organoids from 1, 000 cells each, seed each with (1,200 x 1,000 cells each=) 1.2 x 106 cells. Adjust the cell density in the cell suspension in a way that the cells are seeded in a volume of 0.5 mL of medium. For organoids formed from 1,000 cells each, use a density of 2.4 x 106 cells per mL (1.2 x 106 cells in 0.5 mL).</li>
</ol>
5. Preparation of microwells to receive cells
NOTE: To ensure that the cells do not adhere to the microwell surface, treat with a surfactant rinsing solution that is available for purchase by the manufacturer.
<ol>
	<li>Add 0.5 mL of rinsing solution to each well. Ensure that no air bubbles are trapped in the well. To remove air bubbles, if any, centrifuge the plate at 2,000 x g with brakes at 25 &#176;C for 2 min.</li>
	<li>Observe the plate under a low-magnification inverted microscope, to verify that the bubbles have been removed from the microwells. If trapped bubbles are observed, centrifuge again at 2,000 x g with brakes at 25 &#176;C for 2 min to remove any remaining bubbles.</li>
	<li>Cover the plate with the lid and incubate for 30 min at room temperature. After the treatment is complete, remove the rinsing solution and immediately wash the plate with sterile water or PBS. Ensure that the plate do not dry out after the treatment with the rinsing solution.</li>
</ol>
6. Generation of testicular organoids
<ol>
	<li>Add 0.5 mL of organoid formation medium (without any cells) to each well and centrifuge at 2,000 x g with brakes at 25 &#176;C for 2 min to remove any trapped air bubbles. Observe the well under a low-magnification inverted microscope to verify that the air bubbles have been removed. If trapped bubbles are observed, centrifuge again at 2,000 x g with brakes at 25 &#176;C for 2 min to remove any remaining bubbles.</li>
	<li>Add 0.5 mL of the working cell suspension and gently mix by pipetting up and down. Centrifuge at 500 x g with brakes at 25 &#176;C for 5 min. Use an inverted microscope to verify that the cells have clustered within the microwells.</li>
	<li>Transfer the plate into a cell culture incubator and culture for 5 days. Change half of the medium every other day.
	<ol>
		<li>To change medium without losing any organoids, touch the pipette tip to the wall of the well and lower gently to touch the meniscus and slowly draw the medium. Make sure to follow the meniscus as it drops down. To add fresh medium, touch the pipette tip to the wall of the well on the same side as the medium withdrawal and add fresh medium gently against the well wall, allowing it to slowly flow down the wall.</li>
	</ol>
	</li>
	<li>To recover the organoids, gently pipette the medium up and down using a wide mouth pipette. This would allow the organoids to come out of the microwells.</li>
	<li>Collect the organoids, wash with PBS, fix and perform immunocytochemistry for germ cell marker (UCH-L1), Sertoli cell marker (GATA Binding Protein 4-GATA4), peritubular myoid cell marker (&#945;-Smooth Muscle Actin-&#945;SMA) and Leydig cell marker (Cytochrome P450-CYP450) as described12 and visualize under a confocal microscope.</li>
</ol>

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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Takebe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+and+functional+human+liver+from+an+iPSC-derived+organ+bud+transplant.">Vascularized and functional human liver from an iPSC-derived organ bud transplant.</a> Nature. 499 (7459), 481-484 (2013).</li><li>Quadrato, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+diversity+and+network+dynamics+in+photosensitive+human+brain+organoids.">Cell diversity and network dynamics in photosensitive human brain organoids.</a> Nature. 545 (7652), 48-53 (2017).</li><li>Abbott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture:+biology's+new+dimension.">Cell culture: biology's new dimension.</a> Nature. 424 (6951), 870-872 (2003).</li><li>Pendergraft, S. 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Dr. Ungrin has a financial interest in the AggreWell technology as an inventor.

We have established a simple method that allows the consistent, repeatable generation of large numbers of testicular organoids with tissue architecture that is similar to testis in vivo12. While the approach was developed using porcine testis cells, it is more broadly applicable also to mouse, non-human primate and human testis12. A number of different methods have been reported for producing testicular organoids8,9...

In recent years, there has been a resurgence of interest in three-dimensional (3D) organoids. Different organs such as intestine1, stomach2, pancreas3,4, liver5, and brain6 have been successfully derived into 3D organoid systems. These organoids have architectural and functional similarities to the organs in vivo and are more biologically relevant for study of tissue microenvironment than monolayer culture systems7. As a result, testicular organoids have started to garner interest as well8,9,10,11,12. The majority of methods reported so far are complex, non-high throughput10 and require the addition of ECM proteins8,10. This complexity also leads to issues with reproducibility. A simple and reproducible method is needed that allows for the generation of testicular organoids with cell-associations that are like testis in vivo.
We have recently reported a system to address these requirements12. Using the pig as a model, we employed a centrifugal forced aggregation approach in the microwell system. In the microwell system, each well contains a large number of identical smaller microwells13. This allows for the generation of numerous spheroids of uniform size. The microwell system enabled generation of large numbers of uniform organoids with a testis-specific architecture. The system is simple and does not require addition of ECM proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm ultra low attachment tissue culture dish</td>
			<td>Corning</td>
			<td>#CLS3262</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm tissue culture dish</td>
			<td>Corning</td>
			<td>#353803</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrwell 400</td>
			<td>Stemcell Technologies</td>
			<td>#34411</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>Stemcell Technologies</td>
			<td>#07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type IV from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>#C5138</td>
			<td>referred as Collagenase IV S</td>
		</tr>
		<tr>
			<td>Collagenase type IV Worthington</td>
			<td>Worthington-Biochem</td>
			<td>#LS004189</td>
			<td>referred as Collagenase IV W</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>#DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium/F12</td>
			<td>Gibco</td>
			<td>#11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>#D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>#D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor</td>
			<td>R&#38;D Systems</td>
			<td>#236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell Strainers 70 &#181;m</td>
			<td>FisherScientific</td>
			<td>#352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Cell Strainers 40 &#181;m</td>
			<td>FisherScientific</td>
			<td>#352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>#12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium</td>
			<td>Gibco</td>
			<td>#41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>#P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine testicular tissue</td>
			<td>Sunterra Farms Ltd (Alberta, Canada)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit</td>
			<td>Millipore</td>
			<td>#SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>#T4049</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of porcine testicular organoids with testis specific architecture using microwell culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Isolated cells from 1-week old porcine testes that were cultured in the microwells self-organized into spheroids (Figure 1A, Figure 2), with delineated and distinct exterior (seminiferous epithelium) and interior compartments (interstitium) (Figure 1B, Figure 2). The two compartments were separated by a collagen IV+ve basement...

Watch this Scientific Journal Video about Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture at JoVE.com

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...

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8.7K Views.  University of Calgary. Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

Video: Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

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 ...

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

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.

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 ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...