Name: three-dimensional inflammatory human tissue equivalents of gingiva
Uploaded: 2018-04-03T14:00:00
Description: Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

This work was supported in part by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (26861689 and 17K11813). The authors would like to thank Mr. Nathaniel Green for proofreading.

This protocol is designed to create human gingival tissue equivalents, gingival wound models, and gingivitis models. Human skin epidermal keratinocytes (HaCaT) were kindly provided from Professor Norbert E. Fusenig of Deutsches Krebsforschungszentrum (Heidelberg, Germany)7. Human gingival fibroblasts (HGFs) were isolated from gingival tissues according to the previously published protocols8. Informed consent was obtained beforehand, and the study was approved according to t...

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This protocol is based on methods of creating gingival tissue equivalents and subcutaneous adipose-tissue equivalents described by previous reports8,21,22. Although this is a simple and easy method, some steps require special attention. For example, the collagen mixture should be kept on ice until use to avoid gel formation in the solution. When adding the collagen mixture into the culture insert, make sure the solution was inje...

Periodontal diseases are the leading cause of tooth loss in adults. Gingivitis and periodontitis are the most common periodontal diseases. Both present biofilm-mediated acute or chronic inflammatory changes in gingiva. Gingivitis is characterized by acute inflammation, whereas periodontitis usually presents as chronic inflammation. On the histological level,bacterial components trigger the activation of immune cells, such as macrophages, lymphocytes, plasma cells, and mast cells1,2. These immune cells, especially macrophages, interact with local cells (including gingival epithelial cells, fibroblasts, endothelial cells, and osteoblasts) resulting in inflammatory lesions in periodontal tissue3,4. Experimental models of periodontal diseases have been established in various types of animals, such as rats, hamsters, rabbits, ferrets, canines, and primates. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines of periodontal diseases5. Co-cultivation of periodontal bacteria and monolayer human oral epithelial cells has been used to investigate the mechanism of periodontal infections6. Nevertheless, monolayer cultures of oral cells lack the three-dimensional (3D) cellular architecture of intact tissue; therefore, they cannot mimic the in vitro situation.
Here, 3D inflammatory human tissue equivalents of gingiva (iGTE) are established to represent periodontal diseases in vitro. This 3D model of periodontal diseases occupies an intermediate position between monolayer cell cultures and animal models. Three types of human cells, including HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, are co-cultivated on collagen gel, and stimulated by inflammatory initiators to build iGTE. IGTE closely simulates the in vivo conditions of cell differentiation, cell-cell interaction, and macrophage activation in gingiva. This model has many possible applications for drug screening and testing new pharmacological approaches in periodontal diseases, as well as for analyzing cellular and molecular mechanisms in wound healing, inflammation, and tissue regeneration.

<table>
	<tbody>
		<tr>
			<td>Collagen type I-A</td>
			<td>Nitta Gelatin Inc</td>
			<td></td>
			<td>For making dermis of GTE</td>
		</tr>
		<tr>
			<td>MEM-alpha</td>
			<td>Thermo Fisher Scientific</td>
			<td>11900073</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>Cell Culture Insert (for 24-well plate), pore size 3.0 &#956;m</td>
			<td>Corning, Inc.</td>
			<td>353096</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31600034</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828028</td>
			<td>Cell culture reagent</td>
		</tr>
		<tr>
			<td>Tissue puncher</td>
			<td>Shibata system service co., LTD</td>
			<td>SP-703</td>
			<td>For punching holes in GTE</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>31800022</td>
			<td>Cell culture medium</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A3294</td>
			<td>For immunostaining</td>
		</tr>
		<tr>
			<td>Hoechst 33342 (NucBlue Live Cell stain)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37605</td>
			<td>For labeling nuclei</td>
		</tr>
		<tr>
			<td>Fluorescence mount medium</td>
			<td>Dako</td>
			<td></td>
			<td>For mounting samples after immunostaining</td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin 8+18 antibody [5D3]</td>
			<td>abcam</td>
			<td>ab17139</td>
			<td>For identifying epithelium</td>
		</tr>
		<tr>
			<td>Scaning electron microscope</td>
			<td>Hitachi, Ltd.</td>
			<td>HITACHI S-4000</td>
			<td>For observing samples&#39; surface topography and composition</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscopy</td>
			<td>LSM 700; Carl Zeiss Microscopy Co., Ltd.</td>
			<td>LSM 700</td>
			<td>For observing samples&#39; immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin 19 antibody</td>
			<td>abcam</td>
			<td>ab52625</td>
			<td>For identifying epithelium</td>
		</tr>
		<tr>
			<td>Anti-vimentin antibody</td>
			<td>abcam</td>
			<td>ab92547</td>
			<td>For identifying fibroblasts and activated macrophages</td>
		</tr>
		<tr>
			<td>Anti-TE-7 antibody</td>
			<td>Millipore</td>
			<td>CBL271</td>
			<td>For identifying fibroblasts in the dermis</td>
		</tr>
		<tr>
			<td>Anti-CD68 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>SAB2700244</td>
			<td>For identifying macrophages</td>
		</tr>
		<tr>
			<td>Human CD14 Antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB3832-SP</td>
			<td>For identifying macrophages</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594-conjugated secondary goat anti-rabbit antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11012</td>
			<td>For immunofluorescence staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated secondary goat anti-mouse antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11001</td>
			<td>For immunofluorescence staining</td>
		</tr>
		<tr>
			<td>EVOS FL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>For observing the sample&#39;s immunofluorescence staining</td>
		</tr>
		<tr>
			<td>THP-1 cells</td>
			<td>Riken BRC cell bank</td>
			<td>RCB1189</td>
			<td>For making iGTE</td>
		</tr>
		<tr>
			<td>PMA(Phorbol 12-myristate 13-acetate)</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td>For differentiatting THP-1 cells</td>
		</tr>
	</tbody>
</table>

three-dimensional inflammatory human tissue equivalents of gingiva

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental physiopathology of periodontal diseases. Current experimental models of periodontal diseases have been established in various types of animals. However, the physiopathology of animal models is different from that of humans, making it difficult to analyze cellular and molecular mechanisms and evaluate new medicines for periodontal diseases. Here, we present a detailed protocol for reconstructing human inflammatory tissue equivalents of gingiva (iGTE) in vitro. We first build human tissue equivalents of gingiva (GTE) by utilizing two types of human cells, including human gingival fibroblasts (HGF) and human skin epidermal keratinocytes (HaCaT), under three-dimensional conditions. We create a wound model by using a tissue puncher to punch a hole in the GTE. Next, human THP-1 monocytes mixed with collagen gel are injected into the hole in the GTE. By adimistration of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) for 72 h, THP-1 cells differentiated into macrophages to form inflammatory foci in GTE (iGTE) (IGTE also can be stumilated with 2 &#181;g/mL of lipopolysaccharides (LPS) for 48 h to initiate inflammation). IGTE is the first in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture. IGTE reflects major pathological changes (immunocytes activition, intracellular interactions among fibryoblasts, epithelial cells, monocytes and macrophages) in periodontal diseases. GTE, wounded GTE, and iGTE can be used as versatile tools to study wound healing, tissue regeneration, inflammation, cell-cell interaction, and screen potential medicines for periodontal diseases.

HaCaT cells displayed typical keratinocyte morphology under phase-contrast microscopic observation (Figure 2A). Scanner electron microscopic (SEM) images showed that HaCaT cell surfaces were covered by many microvilli. Intercellular connections between HaCaT cells were mediated by membrane processes (Figure 2B). HaCaT cells expressed gingival epithelium marker K8/1814, indicating that HaCaT cells are suita...

Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

The goal of the protocol is to build an inflammatory human gingiva model in vitro. This tissue model co-cultivates three types of human cells, HaCaT keratinocytes, gingival fibroblasts, and THP-1 macrophages, under three-dimensional conditions. This model can be applied to investigating periodontal diseases, such as gingivitis and periodontitis.

Periodontal diseases (such as gingivitis and periodontitis) are the leading causes of tooth loss in adults. Inflammation in gingiva is the fundamental ...

The overall goal of this experiment is to reconstruct a 3D inflammatory gingiva tissue model by using three types of human cells in vitro. This method can help study wound healing, tissue regeneration, inflammation, and screen potential medicines for periodontal diseases. The main advantage of this technique is that this is the first method to build an in vitro model of inflammatory gingiva using human cells with a three-dimensional architecture.In a 15-milliliter sterile tube, add collagen type I-A, 10%concentrated MEM-alpha, and 10%reconstruction buffer. Then, leave the collagen mixture on ice till the next use. Next, position the 24-well culture inserts with three micrometer pore size in a 24-well culture plate.Then, use 25%trypsin-EDTA solution to detach the HGFs from the culture flask. Next, suspend the detached cells in 10 milliliters of culture medium. Then, count the number of cells using a Burker-Turk cell counter.After isolating the desired cell number, centrifuge the cells at 190 times g for four minutes. Then, suspend the cell pellet with the collagen solution, and leave the tube on ice. Next, add 5 milliliters of cell-collagen mixture into the culture insert without producing any air bubbles.Then, incubate the culture insert with the cell-collagen mixture at 37 degrees Celsius and 5%carbon dioxide for 10 to 30 minutes. After the incubation, add one milliliter of the culture medium into the well and 5 milliliter of the medium into the insert. Then, incubate the culture plate at 37 degrees Celsius with 5%carbon dioxide for a day.Next, use 25%of trypsin-EDTA solution to detach the HaCaT cells from the culture plate. Once the cells are detached, add 10 milliliters of medium B to the cells. Then, count the cell number.After isolating the desired cell number, centrifuge the cells at 190 times g for four minutes. After the centrifugation, dissolve the cell pellet in medium B.Next, aspirate the medium from the culture insert. Then, add 5 milliliters of HaCaT cell suspension over the HGF-collagen mixture.Leave the culture insert in the incubator for a day. The next day, replace the culture medium. Add one milliliter of co-culture KSR medium supplemented with 5%fetal bovine serum into the culture well and 5 milliliter in the culture insert.Then, incubate the culture plate at 37 degrees Celsius for 24 hours. The next day, replace the culture medium with co-culture KSR medium supplemented with 1%fetal bovine serum. Remove the entire culture medium from both the culture insert and the culture well after 24 hours.Then, add fresh 7 to one milliliter of KSR medium in the culture well only, and continue culturing for two weeks. Remember to change the medium in the culture well twice every week. Prepare a one to three millimeter diameter tissue puncher, and sterilize the puncher in the autoclave at 121 degrees Celsius for 20 minutes.After sterilizing, push the tissue puncher perpendicularly into GTE for about 30 micrometers deep to make a wound in the tissue. Next, mix 10 nanograms per milliliter of PMA, 10%reconstruction buffer, and 10%concentrated RPMI medium with collagen type I-A gel. Then, leave the collagen mixture on ice till next use.Next, centrifuge the THP-1 cells at 190 times g for four minutes. After centrifugation, suspend the cell pellet in one milliliter of collagen solution and leave on ice. Then, add 10 to 30 microliters of the THP-1 cell-collagen mixture to the wounded area of the GTE.Next, replace the culture medium with 7 to one milliliter KSR medium containing 10 nanograms per milliliter of PMA. Then, incubate the mixture at 37 degrees Celsius at 5%carbon dioxide for 72 hours. To characterize the reconstructed GTE, H and E staining and scanning electron microscopy are done.The images show GTE exhibiting a gingiva-like tissue structure with multiple layers of epithelium and lamina propria. The images also show that the HGF cells in the reconstructed GTEs present distinct morphological characteristics of fibroblasts surrounded by collagen fibers. Then, immunofluorescence staining is done to study the expression of dermal and epithelial markers.This is to validate if GTE and iGTE have the molecular features of the real inflammatory human gingiva. The images show vimentin and TE-7 are strongly present in the lamina propria of GTE. K19, a specific marker to junctional epithelium in periodontitis, though weakly expresses in the GTE epithelium, is strong enough in iGTE epithelium.Finally, in order to generate the inflammatory foci in iGTE, PMA-treated THP-1 monocytes are injected in the wounded area of the tissue. At 72 hours after PMA treatment, THP-1 cells are differentiated into macrophage-like cells. Whole-mount immunostaining confirmed that these cells express two important macrophage markers, CD14 and vimentin, and formed the inflammatory foci.Once mastered, this technique can be done in three to four weeks if it is performed properly. While attempting this procedure, it's important to remember to avoid contamination. Following this procedure, other methods, like using LPS to stimulate inflammatory responses, can be performed in order to answer additional questions, like how to induce inflammation through the host-biofilm interactions.After its development, this technique paved the way for researchers in the field of inflammation, tissue regeneration, and wound healing to explore the cellular and molecular mechanism and screen potential medicines for periodontal diseases. After watching this video, you should have a good understanding of how to build a 3D inflammatory gingiva tissue model in vitro.

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Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...