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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Isolation and Culture of Primary Untransformed Stromal Cells
<ol>
	<li>Human tissue collection and preparation.
	<ol>
		<li>Obtain specimens of human omentum, 2 cm3, removed during an abdominal surgery, and immediately immerse tissue in RT phosphate-buffered saline (PBS) (Figure 1A). A piece of omentum 3 cm x 2 cm typically yields 1 million primary human mesothelial cells (HPMC) and 200,000 primary human fibroblasts or normal omental fibroblasts (NOF).</li>
		<li>Spin down the PBS the tissue was immersed in at 0.5 x g for 3 min as soon as possible after collection (&#60; 2 hr in PBS) and transfer the tissue to fresh 20 ml PBS in a 50 ml conical tube. Cut up tissue into 5mm3 pieces by mincing with scalpels in a 15 cm diameter, sterile culture dish (Figure 1B).</li>
	</ol>
	</li>
	<li>Primary human mesothelial cell isolation
	<ol>
		<li>To isolate HPMC, transfer the minced tissue into 50 ml conical tubes and wait 1 min for the solid pieces to float to the top (Figure 1C &#38; D). HPMC and red blood cells (RBC) will be present in the liquid on the bottom. Use a pipette to remove the liquid from the bottom of the tube and into a new conical tube. Spin the liquid down at 0.5 x g for 3 min and aspirate the supernatant from the HPMC/RBC pellet.
		<ol>
			<li>Repeat the process three times: add 20 ml PBS to the minced tissue, allow tissue to rise, remove liquid from the bottom with a pipette and into the HPMC/RBC tube, spin down, and remove the supernatant. After all spins are completed and the supernatant is aspirated, the pellet will contain HPMC and RBC.</li>
		</ol>
		</li>
		<li>Plate all cells from the pellet into a 75 cm2 flask in 15 ml of full growth media (DMEM with 10% fetal bovine serum [FBS],1% MEM vitamins [93 mg/L], 1% MEM nonessential amino acids [81.4 mg/L], 1% penicillin streptomycin [Pen-Strep, 100 U/ml penicillin and 100 &#181;g/ml streptomycin]).</li>
		<li>Perform a secondary PBS wash to isolate additional HPMCs.
		<ol>
			<li>For the secondary PBS wash, shake the remaining solid tissue at 200 rpm, 37 &#176;C for 10 min in 20 ml of PBS, then wait 1 min for the tissue to float to the top. Remove the liquid from the bottom of the tube into a new tube, centrifuge at 0.5 x g for 3 min, and aspirate the supernatant.</li>
			<li>Plate all the HPMC/RBC from the secondary PBS wash in a separate 75&#160; cm2 flask in 15 ml of full growth media.</li>
		</ol>
		</li>
		<li>To isolate any remaining HPMC, shake the minced tissue at 200 rpm, 37 &#176;C degrees for 10 min in 20 ml of a 1:1 0.25% trypsin 25 mM EDTA:PBS solution by volume. Allow the tissue to rise, collect the liquid at the bottom of the tube into a new 50 ml tube, and spin down at 0.5 x g for 3 min.
		<ol>
			<li>Aspirate the supernatant and plate all the HPMC/RBC in a separate 75 cm2 flask in 15 ml of full growth media.</li>
		</ol>
		</li>
		<li>Culture the plated cells at 37&#176;C and 5% CO2 in a humidified environment. Feed plated cells by adding 15 ml of full growth media on days 3 and 5 without removing the spent media. HPMC can be cultured for 5-7 days before splitting.
		<ol>
			<li>Use low-passage HPMC (up to passage 2) in all experiments to minimize dedifferentiation and modification of original phenotype. Use a wide-field microscope preferably with a 20x objective with plastic differential interference contrast capabilities or 4-20x objective with phase contrast capabilities (phase contrast filters on microscope) for taking all images of cells, and a 10x objective ()in bright field to analyze immunohistochemical 3,3&#39;-Diaminobenzidine (DAB) staining.</li>
		</ol>
		</li>
		<li>Confirm that the HPMC are cuboidal and express cytokeratin 8 and vimentin by performing immunohistochemistry (Figure 2A, C, E).</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>PBS</td><td>Fisher Scientific</td><td>SH3001304</td><td></td></tr><tr><td>Single-edged razor blades</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>15 cm culture dishes</td><td>BD Biosciences</td><td>353025</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000044_3616914956</td><td></td></tr><tr><td>DMEM with L-Glutamine</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>MEM Vitamins</td><td>Corning</td><td>25-020-Cl</td><td></td></tr><tr><td>MEM Nonessential amino acids</td><td>Corning</td><td>25-025-CI</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Corning</td><td>30-002-CI</td><td></td></tr><tr><td>Shaker</td><td>Thermo-Fisher</td><td>MaxQ 4450</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Incubator</td><td>Thermo-Fisher</td><td>Forma Series II Water Jacketed CO2 Incubator Model 3100</td><td></td></tr><tr><td>T-75 Flasks</td><td>BD Biosciences</td><td>353136</td><td></td></tr><tr><td>T-175 Flasks</td><td>BD Biosciences</td><td>353112</td><td></td></tr><tr><td>Pipet tips</td><td>Rainin</td><td>P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Axiovert 200m</td><td></td></tr><tr><td>Anti-Integrin &amp;alpha;V&amp;beta;3 Antibody, clone LM609</td><td>EMD Millipore</td><td>MAB1976</td><td></td></tr><tr><td>Beta 1</td><td>Oncosynergy</td><td>OS2966</td><td></td></tr><tr><td>Alpha 5 [CD49e]</td><td>ID Pharmingen</td><td>555615</td><td></td></tr><tr><td>Beta 4 [CD104]</td><td>EMD Millipore</td><td>MAB 2058</td><td></td></tr><tr><td>Pipet tips</td><td>Corning</td><td>Filtered tips P2, P10, P20, P200 and P1000</td><td></td></tr><tr><td>Glass flask</td><td></td><td></td><td></td></tr></tbody></table>

mesothelial cell isolation: a technique to isolate primary mesothelial cells from human omentum tissue specimen

Source: Peters, P. N. et al. <a href="https://jove.unicartagenaproxy.elogim.com/t/53541">Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity.</a> J. Vis. Exp. (2015)
This video describes the protocol to isolate primary human mesothelial cells from the human omentum specimen. The mesothelial cells can be used to establish an in vitro ovarian cancer model to investigate ovarian cancer cell adhesion, invasion, and proliferation.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/20376/20376_Figure1.jpg" /> 
Figure 1. Isolation of primary human omental cells. (A) A piece of omentum is collected at surgery in PBS and transported to the laboratory. The tissue is pelleted at 0.5 x g x 3 min and transferred to fresh PBS. (B) The piece of omentum is transferred to a 10 cm petri dish, washed with PBS, and scalpels are used to cut t...

Mesothelial Cell Isolation: A Technique to Isolate Primary Mesothelial Cells From Human Omentum Tissue Specimen

Source: Peters, P. N. et al. Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity. J. Vis. ...

Human primary mesothelial cells, or HPMCs, line many visceral organs like the omentum or the layer of fatty tissue present in the peritoneal cavity.
To isolate the mesothelial cells, begin by immersing a human omentum specimen in a suitable buffer to remove the excess red blood cells or RBCs. Next, place the omentum in a glass dish and mince it into pieces. This dissociates the tissue, releasing HPMCs along with the contaminating RBCs.
Transfer the pieces into a conical tube and allow them to remain still for the desired time. The minced tissue rich in low-density fat rises to the top, while the dense cellular components settle at the bottom. Next, aspirate the liquid at the bottom, containing HPMCs and RBCs, into a fresh tube.
Centrifuge to collect the cells. Remove the buffer-containing supernatant. Resuspend the pellet in a mesothelial growth medium and incubate for the desired duration. The media facilitates the selective growth of HPMCs over RBCs.
Observe the cells using a light microscope to confirm the growth of cuboidal-shaped human mesothelial cells.

mesothelial-cell-isolation-technique-to-isolate-primary-mesothelial

Universidad de Cartagena UDEC

Research

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Assays & techniques

1.6K ビュー. 出典:Peters, P. N. et al. ヒト腹腔の器官型培養における卵巣がん播種の初期段階をモデル化する。J. Vis. Exp.(2015年) このビデオでは、ヒト大網標本から初代ヒト中皮細胞を単離するプロトコールについて説明しています。中皮細胞は、卵巣がん細胞の接着、浸潤、および増殖を調査するためのin vitro卵巣がんモデルを確立するために使用できます。 

ビデオ: 中皮細胞の単離:ヒト大網組織標本から初代中皮細胞を単離する技術 - 概念

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

黒色腫切片 3 D 回転楕円体の皮膚モデル

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

JoVE Journal

がん研究

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

マウス実験転移性乳癌癌モデルを生成、およびマウス乳癌根治術を実行

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

医学

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

膜成熟性アディポサイト凝集培養(MAAC)を用いたヒト成熟性の単離と培養

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

Mesothelial Cell Isolation: A Technique to Isolate Primary Mesothelial Cells From Human Omentum Tissue Specimen

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Mesothelial Cell Isolation: A Technique to Isolate Primary Mesothelial Cells From Human Omentum Tissue Specimen