Name: three-dimensional inflammatory human tissue equivalents of gingiva
Uploaded: 2018-04-03T14:00:00.000+00:00
Duration: 8 min 43 s
Description: Watch this Scientific Journal Video about Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva at JoVE.com

이 작품 지원 부분에서 일본 사회에 의해 과학 진흥 (JSP) 선진적인 과학 연구 (26861689 및 17 K 11813) 한다. 저자 나 다니엘 그린 씨 교정 하는 것을 감사 하 고 싶습니다.

이 프로토콜은 인간의 gingival 조직 등가물, gingival 상처 모델 및 치은 염 모델을 만들 하도록 설계 되었습니다. 인간의 피부 상피 keratinocytes (HaCaT) 교수 노 버트 E. Fusenig 독일 Krebsforschungszentrum (하이 델 베르크, 독일)7의에서 친절 하 게 제공 되었다. 인간의 gingival 섬유 아 세포 (HGFs)8이전 게시 된 프로토콜 따라 gingival 조직에서 분리 했다. 미리 동의 얻어와 연구 윤리 위원회는 일본 치과 대학 학교 생활 치과의 도쿄에서 설정한 지침에 따라 승인 (승인 번호: NDU-T2012-35). 1-3 단계 프로토콜 셀 문화 후드에서 수행 되어야 합니다.
1. 3D 인체 조직 등가물 Gingiva (GTE) (그림 1A)의 준비
<ol><li>10% 혼합 콜라겐 타입-A의 젤 집중 MEM-알파와 10% 재건 버퍼 (2.2 g NaHCO3 및 4.47 g HEPES 100 ml 0.05 N NaOH) pipetting으로 15 mL 무 균 튜브에. 사용 될 때까지 얼음에 혼합된 콜라겐 솔루션을 유지.</li>
	<li>24 잘 문화 삽입 (기 공 크기 3.0 µ m) 24-잘 접시에 놓습니다.</li>
	<li>
0.25% trypsin EDTA 용액을 사용 하 여 문화 표면에서 HGFs를 제거 합니다. 10 ml 문화 매체 A의 세포를 중단 (MEM-알파 + 20 %FBS + 1 %GlutaMAX). Bürker-터크 셀 카운터로 셀을 계산.
	<ol>
<li>셀 (1-5 x105 셀/mL), 원하는 수량을 추출 후 4 분 190 x g에서 원심.</li>
		<li>혼합된 콜라겐 솔루션에 셀을 일시 중단 합니다. 다음 단계까지 얼음에 혼합물을 유지.</li>
	</ol>
</li>
	<li>모든 거품을 생산 하지 않고 문화 삽입으로 셀 콜라겐 혼합 (0.5-2.5 x 105 셀/잘) 0.5 mL를 추가 합니다. 젤에 혼합물을 응고를 37 ° C에서 5% CO2 습도 분위기에서 10-30 분을 위한 혼합물을 품 어. 참고:의 문화 삽입 되지 않도록 불평등 콜라겐 젤을 중심으로 콜라겐 혼합물을 추가 합니다.</li>
	<li>잘 삽입으로 0.5 mL으로 문화 매체의 1 mL를 추가 합니다. 24 h에 대 한 문화 접시를 품 어 또는 37 ° c.에 5% CO2 습도 분위기에서 하룻밤</li>
	<li>0.25% trypsin EDTA 용액을 사용 하 여 문화 표면에서 HaCaT 세포를 제거 합니다. 10 ml 매체 B의 셀을 일시 중단 (DMEM + 10 %FBS + 1 %GlutaMAX) 셀을 카운트. 추출 셀 (1-5 x5 셀/10ml) 피 펫, 190 x g.에서 4 분에 대 한 원심 분리기의 필요한 수량 중단 중간 b 세포</li>
	<li>열망 하 여 HGF 콜라겐 혼합물을 포함 하는 문화 삽입에서 매체를 제거 합니다. HGF 콜라겐 혼합 상단 HaCaT 세포 현 탁 액 (0.5-2.5 x 105 셀/잘) 0.5 mL를 추가 합니다. 24 시간 또는 하룻밤에 대 한 문화를 품 어. 참고:이 시간이 시점에서 문화에 매체 잘 해야 중간 A 문화 삽입에 중간 중간 B. 해야 하는 동안</li>
	<li>KSR 매체 (공동 문화 매체)와 문화 매체를 대체 (메모리 알파 + 15% 녹아웃 혈 청 교체 + 1 %GlutaMAX) + 5 %FBS. 잘 하 고 문화 삽입으로 0.5 mL 문화에 문화 매체의 1 mL를 추가 합니다. 24-48 h에 대 한 문화를 품 어.</li>
	<li>KSR 매체 + 1% 문화 매체를 대체 FBS. 잘 문화에 1 mL 및 문화 삽입으로 0.5 mL를 추가 합니다. 24 시간 또는 하룻밤에 대 한 문화를 품 어.</li>
	<li>문화에서 문화 매체 0.7-1 잘 바꿉니다 mL KSR 매체. (문화 표면 공기에 노출 한다) 문화 삽입에서 매체를 완전히 제거 합니다. 1-2 주에 대 한 문화를 품 어. 일주일에 2 번 잘 문화에서 매체를 변경 합니다. 참고: (상위 레이어 구성 keratinocytes) 문화 표면 위의 중간 수준 상승을 못하게 합니다. 항상 문화 표면 건조 유지.</li>
</ol>2. 부상된 GTE (그림 1B)의 준비
<ol><li>1-3 m m 직경 조직 주먹을 준비 합니다. 20 분 동안 121 ° C에 압력솥으로 소독.</li>
	<li>밀어 조직 주먹 수직 GTE 300 µ m 깊이 대 한 그리고 상처를 시뮬레이션 하기 위해 조직에 구멍을 펀치.</li>
	<li>이 단계에서 부상된 GTE를 사용 하 여 상처 치유 및 조직 재생을 조사. 또는, GTE 조직학 분석을 수행 하기 위해 10% 포 르 말린 중립 버퍼 솔루션을 사용 하 여 수정 합니다.</li>
</ol>3. 염증 GTE (iGTE) (그림 1B)의 준비
<ol><li>이전 보고서9에 따르면 THP-1 세포 배양.</li>
	<li>10% 혼합 콜라겐 타입-A의 젤 집중 RPMI 1640, 10% 재건 버퍼 (2.2 g NaHCO3 및 4.47 g HEPES 100 ml 0.05 N NaOH)과 10 ng/mL PMA. 사용까지 얼음에 혼합된 콜라겐 솔루션을 유지.</li>
	<li>수 1-2 x 106 THP-1 세포와 190 x g.에서 4 분 동안 원심 분리기 혼합된 콜라겐 솔루션의 1 mL에 셀을 일시 중단 합니다. 다음 단계까지 얼음에 혼합물을 유지.</li>
	<li>GTE의 부상된 지역으로 10-30 µ L THP-1-콜라겐 혼합물을 추가 합니다. 0.7-1 문화 매체를 대체 PMA의 10 ng/mL를 포함 하는 mL KSR 매체.</li>
	<li>5% CO2 72 h에 대 한 37 ° C에서 습도 분위기에서 혼합물을 품 어.</li>
	<li>치은 염 이나 치 주 질환 조사에 대 한 모델을 사용 하 여 (예를 들어 추가 잠재적인 항 염증 제 약 cytokine 릴리스10에 그것의 효과 볼 수 iGTE). 또는, 72 h PMA 처리를 생략 2 µ g/mL LPS의 문화에 추가 하 고 조직 5% CO2 48 h11,1237 ° C에서 습도 분위기에서 품 어.</li>
</ol>4. 고정 및 전체 마운트 Immunostaining GTE와 iGTE의 문화
<ol><li>
문화는 고정
	<ol>
<li>24-잘 접시에서 문화 매체를 제거 합니다. 워시 버퍼 인산 염 (PBS)와 2 회 조직.</li>
		<li>문화를 잘 10% 포 르 말린 중립 버퍼 솔루션 삽입 하 고 1 mL의 1 mL를 추가 합니다. 하룻밤 냉장고에서 4 ° C에 24-잘 접시를 남겨 주세요.</li>
		<li>다음 날 10% 포 르 말린 중립 버퍼 솔루션을 제거 합니다.</li>
		<li>전체 산 immunostaining에 대 한 고정 후 PBS로 3-4 회 조직을 씻어.</li>
		<li>되며 오신 (H & E) 얼룩 및 일반 immunostaining, 탈수, 파라핀 포함, 및 단면 진행. 참고: 전체 산 immunostaining에 대 한이 단계를 생략할 수 있습니다.</li>
	</ol>
</li>
	<li>
전체 마운트 immunostaining 참고:이 프로토콜 참조 13에서에서 수정 되었습니다.<ol>
<li>3 조직 세척 PBS 0.5-1에서에서 x % 트리톤, 10-30 분 각 시간.</li>
		<li>블로킹 버퍼에서 30 분 동안 두 번 조직을 품 어 (PBS 1% 트라이 톤 + 10 %BSA), 실내 온도에.</li>
		<li>블로킹 버퍼, 5-10 분 때마다 조직 2 ×를 씻어.</li>
		<li>삽입에 필요한 희석/농도에서 1 차적인 항 체 솔루션의 50 µ L를 추가 합니다. 얇은 플라스틱 집착 영화를 사용 하 여 조직에 밖으로 건조 하지 않도록 하려면 삽입 하.</li>
		<li>4 ° c.에 1 ~ 2 일에 대 한 조직을 품 어</li>
		<li>씻어 조직 3 번, PBS에서 30 분 1% 트라이 톤 + 10 %BSA. 씻어 PBS에서 5 분, 3 번 다시 1% 트라이 톤.</li>
		<li>블로킹 버퍼에 이차 항 체의 50 µ L를 추가 (PBS 1% 트라이 톤 + 10 %BSA) 및 각 삽입을 Hoechst 33342 솔루션 (NucBlue 라이브 셀 얼룩)의 5 µ L. 얇은 플라스틱 집착 영화를 사용 하 여 조직에 밖으로 건조 하지 않도록 하려면 삽입 하.</li>
		<li>4 ° c.에 1 ~ 2 일 동안 품 어 젖은 종이 타월에 24-잘 접시를 포장 하 고 보육 전반에 걸쳐 사용을 비닐 팩에 넣어.</li>
		<li>PBS 1% 트라이 톤에서 10 분 조직 3 번 씻어</li>
		<li>날카로운 바늘을 사용 하 여 얻을 조직 문화 삽입의 막 잘라. 장소는 슬라이드에 막입니다. 참고: 조직 막에 있어야 한다.</li>
		<li>형광 마운트 매체의 2-3 방울을 추가 합니다. 커버 글라스와 어둠 속에서 4 ° C에서 저장 조직 분석까지 커버.</li>
		<li>Confocal 레이저 스캐닝 현미경 (LSM) 조직 보기</li>
	</ol>
</li>
</ol>

<ol>
	<li>Cekici, A., Kantarci, A., Hasturk, H., Van Dyke, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+and+immune+pathways+in+the+pathogenesis+of+periodontal+disease.">Inflammatory and immune pathways in the pathogenesis of periodontal disease.</a> Periodontology 2000. 64 (1), 57-80 (2014).</li><li>Hasturk, H., Kantarci, A., Van Dyke, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+Inflammatory+Diseases+and+Systemic+Inflammation:+Role+of+the+Macrophage.">Oral Inflammatory Diseases and Systemic Inflammation: Role of the Macrophage.</a> Frontiers in Immunology. 3, 118 (2012).</li><li>Koh, T. J., DiPietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+wound+healing:+The+role+of+the+macrophage.">Inflammation and wound healing: The role of the macrophage.</a> Expert reviews in molecular medicine. 13, e23 (2011).</li><li>Mescher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+fibroblasts+during+inflammation+and+tissue+repair+in+models+of+organ+regeneration.">Macrophages and fibroblasts during inflammation and tissue repair in models of organ regeneration.</a> Regeneration. 4 (2), 39-53 (2017).</li><li>Struillou, X., Boutigny, H., Soueidan, A., Layrolle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Animal+Models+in+Periodontology:+A+Review.">Experimental Animal Models in Periodontology: A Review.</a> The Open Dentistry Journal. 4, 37-47 (2010).</li><li>Han, Y. W., 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=Interactions+between+Periodontal+Bacteria+and+Human+Oral+Epithelial+Cells:+Fusobacterium+nucleatum+Adheres+to+and+Invades+Epithelial+Cells.">Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells.</a> Infection and Immunity. 68 (6), 3140-3146 (2000).</li><li>Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., Fusenig, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+keratinization+in+a+spontaneously+immortalized+aneuploid+human+keratinocyte+cell+line.">Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.</a> J Cell Biol. 106 (3), 761-771 (1988).</li><li>Xiao, L., Miwa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen-rich+water+achieves+cytoprotection+from+oxidative+stress+injury+in+human+gingival+fibroblasts+in+culture+or+3D-tissue+equivalents,+and+wound-healing+promotion,+together+with+ROS-scavenging+and+relief+from+glutathione+diminishment.">Hydrogen-rich water achieves cytoprotection from oxidative stress injury in human gingival fibroblasts in culture or 3D-tissue equivalents, and wound-healing promotion, together with ROS-scavenging and relief from glutathione diminishment.</a> Hum Cell. 30 (2), 72-87 (2017).</li><li>Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., Tada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+human+acute+monocytic+leukemia+cell+line+(THP-1).">Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).</a> Int J Cancer. 26 (2), 171-176 (1980).</li><li>Ara, T., Kurata, K., Hirai, K., Uchihashi, T., Uematsu, T., Imamura, Y., Furusawa, K., Kurihara, S., Wang, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gingival+fibroblasts+are+critical+in+sustaining+inflammation+in+periodontal+disease.">Human gingival fibroblasts are critical in sustaining inflammation in periodontal disease.</a> J Periodontal Res. 44 (1), 21-27 (2009).</li><li>Park, E. K., Jung, H. S., Yang, H. I., Yoo, M. C., Kim, C., Kim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+THP-1+differentiation+is+required+for+the+detection+of+responses+to+weak+stimuli.">Optimized THP-1 differentiation is required for the detection of responses to weak stimuli.</a> Inflamm Res. 56 (1), 45-50 (2007).</li><li>Sharif, O., Bolshakov, V. N., Raines, S., Newham, P., Perkins, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+the+LPS+induced+NF-kappaB+response+in+macrophages.">Transcriptional profiling of the LPS induced NF-kappaB response in macrophages.</a> BMC Immunol. 8, 1 (2007).</li><li>Shetty, S., Gokul, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinization+and+its+disorders.">Keratinization and its disorders.</a> Oman Med J. 27 (5), 348-357 (2012).</li><li>Klinge, B., Matsson, L., Attstr&#246;m, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+initial+gingivitis+in+humans.+A+pilot+study.">Histopathology of initial gingivitis in humans. A pilot study.</a> J Clin Periodontol. 10 (4), 364-369 (1983).</li><li>Nagarakanti, S., Ramya, S., Babu, P., Arun, K. V., Sudarsan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+E-cadherin+and+cytokeratin+19+and+net+proliferative+rate+of+gingival+keratinocytes+in+oral+epithelium+in+periodontal+health+and+disease.">Differential expression of E-cadherin and cytokeratin 19 and net proliferative rate of gingival keratinocytes in oral epithelium in periodontal health and disease.</a> J Periodontol. 78 (11), 2197-2202 (2007).</li><li>Goodpaster, T., Legesse-Miller, A., Hameed, M. R., Aisner, S. C., Randolph-Habecker, J., Coller, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immunohistochemical+method+for+identifying+fibroblasts+in+formalin-fixed,+paraffin-embedded+tissue.">An immunohistochemical method for identifying fibroblasts in formalin-fixed, paraffin-embedded tissue.</a> J Histochem Cytochem. 56 (4), 347-358 (2008).</li><li>Langeland, K., Rodrigues, H., Dowden, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodontal+disease,+bacteria,+and+pulpal+histopathology.">Periodontal disease, bacteria, and pulpal histopathology.</a> Oral Surg Oral Med Oral Pathol. 37 (2), 257-270 (1974).</li><li>Holness, C. L., Simmons, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+CD68,+a+human+macrophage+marker+related+to+lysosomal+glycoproteins.">Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins.</a> Blood. 81 (6), 1607-1613 (1993).</li><li>Mor-Vaknin, N., Punturieri, A., Sitwala, K., Markovitz, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vimentin+is+secreted+by+activated+macrophages.">Vimentin is secreted by activated macrophages.</a> Nat Cell Biol. 5 (1), 59-63 (2003).</li><li>Xiao, L., Aoshima, H., Saitoh, Y., Miwa, N. <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+squalane-dissolved+on+adipogenesis-accompanied+oxidative+stress+and+macrophage+in+a+preadipocyte-monocyte+co-culture+system.">The effect of squalane-dissolved on adipogenesis-accompanied oxidative stress and macrophage in a preadipocyte-monocyte co-culture system.</a> Biomaterials. 31 (23), 5976-5985 (2010).</li><li>Xiao, L., Aoshima, H., Saitoh, Y., Miwa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+hydroxylated+fullerene+localizes+at+the+cytoskeleton+and+inhibits+oxidative+stress+in+adipocytes+and+a+subcutaneous+adipose-tissue+equivalent.">Highly hydroxylated fullerene localizes at the cytoskeleton and inhibits oxidative stress in adipocytes and a subcutaneous adipose-tissue equivalent.</a> Free Radic Biol Med. 51 (7), 1376-1389 (2011).</li></ol>

저자는 충돌의 관심을 선언합니다.

이 프로토콜 gingival 조직 등가물 및 이전 보고서8,,2122에 의해 설명 된 피하 지방 조직 등가물을 만드는 방법을 기반으로 합니다. 이 간단 하 고 쉬운 방법 이지만, 일부 단계 특별 한 주의가 필요 합니다. 예를 들어 콜라겐 혼합 솔루션에서 젤 형성을 피하기 위해 사용 될 때까지 얼음에 유지 되어야 한다. 문화 삽입으로 콜라겐 ?...

치 주 질환 성인에서 치아 손실의 주요 원인입니다. 치은 염 및 치 주 염 가장 일반적인 치 주 질병이 있습니다. 둘 다 gingiva에 biofilm 중재 급성 또는 만성 염증 성 변화를 제시. 치은 염 치 주 염이 만성 염증으로 일반적으로 선물 하는 반면 급성 염증, 특징 이다. 조직학 수준에 세균성 구성 요소는 대 식 세포, 림프 톨, 플라스마 세포, 돛대 세포1,2등 면역 세포의 활성화를 트리거합니다. 이러한 면역 세포 특히 대 식 세포, 지방 세포 (gingival 상피 세포, 섬유 아 세포, 내 피 세포, 및 osteoblasts 포함)와 상호 작용 결과로 치 주 조직3,4에 염증 성 병 변. 치 주 질환의 실험 모델 동물, 쥐, 등의 다양 한 종류에서 설립 되어 햄스터, 토끼, 흰 족제비, 개, 그리고 대주교. 그러나, 동물 모델의 physiopathology는 인간의 세포 및 분자 메커니즘을 분석 하 고 치 주 질환5의 새로운 의약품을 평가 하기가 어렵습니다 다릅니다. 치 주 박테리아와 단층 인간 구강 상피 세포의 공동 경작은 치 주 감염6의 메커니즘을 조사 하기 위해 사용 되었습니다. 그럼에도 불구 하 고, 구강 세포의 단층 문화 부족 그대로 조직;의 3 차원 (3D) 셀룰러 아키텍처 따라서, 그들은 체 외에 상황을 모방 수 없습니다.
여기, 3D 염증 인간의 조직 등가물 gingiva (iGTE)의 생체 외에서치 주 질환을 대표 설정 됩니다. 치 주 질환의이 3D 모델 단층 세포 배양 및 동물 모델 사이의 중간 위치를 차지 하고있다. 세 가지 유형의 HaCaT keratinocytes, gingival fibroblasts 및 THP-1 세포를 포함 하 여 인간 셀 공동 콜라겐 젤에 재배 있으며 iGTE 구축 염증 초기자에 의해 자극. IGTE는 밀접 하 게 세포 분화, 세포 세포 상호 작용 및 gingiva에 대 식 세포 활성화의 비보에 조건을 시뮬레이션합니다. 이 모델은 마약 검사 및 치 주 질환, 새로운 약리학 접근을 테스트 뿐만 아니라 상처 치유, 염증, 및 조직 재생에 세포질이 고 분자 메커니즘 분석을 위한 많은 가능한 응용.

<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 &amp;mu;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&#039; 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&#039; 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&amp;amp;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&#039;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

치 주 질환 (치은 염 등 치 주 염)는 성인에서 치아 손실의 주요 원인. Gingiva에 염증 치 주 질환의 근본적인 physiopathology입니다. 치 주 질환의 현재 실험 모델 동물의 다양 한 종류에 설립 되었습니다. 그러나, 동물 모델의 physiopathology는 인간의 세포 및 분자 메커니즘을 분석 하 고 치 주 질환에 대 한 새로운 의약품을 평가 하기가 어렵습니다 다릅니다. 여기, 우리 인간의 염증 성 조직 등가물 gingiva (iGTE) 체 외에서의 재구성에 대 한 자세한 프로토콜 제시. 우리는 먼저 인간의 gingival 섬유 아 세포 (HGF) 등 3 차원 조건 하에서 인간의 피부 상피 keratinocytes (HaCaT), 인간 세포의 두 종류를 이용 하 여 gingiva (GTE)의 인간의 조직에 해당 빌드. 우리는 GTE에 구멍을 펀치 조직 주먹을 사용 하 여 상처 모델을 만듭니다. 다음, 인간의 THP 1 monocytes 콜라겐 젤과 혼합는 GTE에 구멍에 주입 됩니다. 10 ng/mL phorbol myristate 12 13-아세테이트 (PMA) 72 h에 대 한 adimistration에 의해 THP-1 셀 GTE (iGTE)에서 양식 염증 foci 하 대 식 세포로 분화 (IGTE 또한 일 수 있다 stumilated lipopolysaccharides (LPS)의 2 µ g/mL와 염증을 시작 하 48 h에 대 한 ). IGTE 3 차원 구조와 인간 세포를 사용 하 여 염증 gingiva의 첫 번째 시험관에 모델입니다. IGTE 치 주 질환에 주요 학적 변화 (immunocytes activition, fibryoblasts, 상피 세포, monocytes 그리고 대 식 세포 중에서 세포 상호 작용)을 반영 한다. 치 주 질환에 대 한 상처 치유 공부를 다양 한 도구, 조직 재생, 염증, 세포 세포 상호 작용, 및 화면 잠재적인 약으로 GTE, 부상된 GTE, 및 iGTE을 사용할 수 있습니다.

HaCaT 세포 단계 대조 현미경 관찰 (그림 2A)에서 전형적인 keratinocyte 형태 표시. 전자 현미경 (SEM) 이미지 스캐너 HaCaT 세포 표면 많은 microvilli에 의해 덮여 있었다 보였다. HaCaT 세포 사이의 세포 연결 막 프로세스 (그림 2B)에 의해 중재 했다. HaCaT 세포 gingival 상피 마커 K8/1814, HaCaT 세포 적합 gingiva 재건 (<strong class="...

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

프로토콜의 목적은 염증 인간의 gingiva 모델 시험관을 구축 하는 것입니다. 이 조직 모델 공동 세 가지 유형의 인간 세포, HaCaT keratinocytes, gingival fibroblasts 및 THP 1 대 식 세포, 3 차원 조건 하에서 양성. 치은 염 및 치 주 염 등 치 주 질환을 조사 하는 것이 모델을 적용할 수 있습니다.

3 차원 선 동적인 인간 조직 Gingiva 등가물

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

three-dimensional-inflammatory-human-tissue-equivalents-of-gingiva

Research

JoVE Journal

Developmental Biology

9.7K 조회수.  The Nippon Dental University. 프로토콜의 목적은 염증 인간의 gingiva 모델 시험관을 구축 하는 것입니다. 이 조직 모델 공동 세 가지 유형의 인간 세포, HaCaT keratinocytes, gingival fibroblasts 및 THP 1 대 식 세포, 3 차원 조건 하에서 양성. 치은 염 및 치 주 염 등 치 주 질환을 조사 하는 것이 모델을 적용할 수 있습니다.

비디오: Three-dimensional Inflammatory Human Tissue Equivalents of Gingiva

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

인간의 3 차원의 조직 공학<em&gt; 시험관</em&gt; 종양 테스트 시스템

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

연구

생체공학

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

쥐과 잇몸 세포의 분리, 처리 및 분석

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

면역학 및 감염병학

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

3 차원 조직 공학 인간의 식도 점막 모델의 제조, 특성 및 사용 가능성

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

세포 외 매트릭스 내에서 임베디드 공학 3 차원 상피 조직

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

절연, 특성화 및 잇몸 면역 세포 네트워크의 기능 시험

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

섬유아세포, 선별 애플리케이션을 위한 인간 공학형 결합 조직

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

새로운 접근 방식을 접목 Neovascularization 인간의 Gingiva에 모니터링

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

의학

Organotypic Tissue Model Systems for Investigating Host-Pathogen Interactions In Vitro

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While in-house grown 2D epithelial monolayers lack true representation of the in vivo situation, commercially available tissue models are often overlooked due to their cost and practicality. However, with careful planning, such models provide reproducible platforms for a vast array of different applications. Here, we report the use of epithelial models that can be utilized for a wide variety of experimental purposes to investigate host-pathogen interactions in various ecological niches, such as the oral cavity, skin, and vaginal mucosa. From simple planktonic cells to complex biofilm co-culture, epithelial models are used to assess microbial adherence and invasion, and to evaluate the host response at a transcriptional and/or protein level, with scope for more detailed profiling using different omics approaches. Furthermore, these biological systems can be used as more accurate test beds for evaluating conventional and novel antimicrobial activity in a complex host-pathogen microenvironment in vitro. The protocols described herein document how models are handled upon arrival and prepared in the laboratory for co-culture stimulation with biofilm communities. The methods detail how experimental outputs are achieved from the model systems, including the processing of tissue, the co-culture setup, and data generation. These experiments include host gene expression through single- and multiplex qPCR analyses and inflammatory protein detection using ELISAs. In conclusion, epithelial models provide useful in vitro systems for preclinical investigatory studies into simple or complex host-pathogen interactions.

The present protocol outlines the application of organotypic tissue models for investigating host-pathogen interactions in vitro. Specifically, the model system described uses a multi-layered oral epithelium exposed to microorganisms associated with biofilms (dental plaque) within the context of oral health versus disease.

In vitro에서 숙주-병원체 상호 작용을 조사하기 위한 유기형 조직 모델 시스템

Recapitulating the host-pathogen interface at the epithelial or mucosal barrier in vitro remains a challenging prospect for infection biologists. While ...

새로운 구강 재갈을 사용하여 Pulp Inflammation Mouse Model 만들기 

Establishment of a Murine Pulp Exposure Model with a Novel Mouth-Gag for Pulpitis Research

Pulpitis, a common cause of natural tooth loss, leads to necrosis and loss of bioactivity in the inflamed dental pulp. Unraveling the mechanisms underlying pulpitis and its efficient treatment is an ongoing focus of endodontic research. Therefore, understanding the inflammatory process within the dental pulp is vital for improving pulp preservation. Compared to other in vitro experiments, a murine pulpitis model offers a more authentic and genetically diverse context to observe the pathological progression of pulpitis. However, using mice, despite their cost-effectiveness and accessibility, poses difficulties due to their small size, poor coordination, and low tolerance, complicating intraoral and dental procedures. This protocol introduces a novel design and application of a mouth-gag to expose mouse pulp, facilitating more efficient intraoral procedures. The mouth-gag, comprised of a dental arch readily available to most dentists and can significantly expedite surgical preparation, even for first-time procedures. Micro-CT, hematoxylin-eosin (HE) staining, and immunofluorescence staining were used to identify changes in morphology and cell expression. The aim of this article is to help researchers establish a more reproducible and less demanding procedure for creating a pulp inflammation model using this novel mouth-gag.

저자 스포트라이트: 개선된 마우스 모델을 통한 치과용 펄프 연구 강화

This article presents a streamlined protocol for establishing a pulpitis model in mice using an innovative mouth-gag, followed by subsequent histological analysis.

3 차원 선 동적인 인간 조직 Gingiva 등가물

요약

더 많은 비디오 탐색

이 비디오의 챕터

인간의 3 차원의 조직 공학