This work was supported by Grants-in-Aid for Scientific Research (KAKENHI) (26461185 and 25460137).

참고 :이 연구는 적절한 윤리위원회에 의해 승인되었다. 인간의 폐 섬유 아 세포의 1 차 문화 <ol><li> 폐 조직의 컬렉션 : <ol><li> 직접 수술 수술실에서 인간의 폐 조직 샘플을 얻습니다. 최대한 멀리 종양에서 수집 비 암성 샘플, 암성 및 비 암성 폐 조직으로부터 약 1cm 3 블록 모아서. </li><li> 둘 베코 변형 이글 배지 100 단위 / ㎖ 페니실린, 100 ㎍ / ml의 스트렙토 마이신 및 0.25 ㎍ / ml의 암포 테리 신 B (무 혈청 배지)로 보충 (DMEM)에서 샘플을 중단. 멸균 50 ㎖ 튜브에 일시 중단 된 샘플을 유지하고 실험실로 전송합니다. 주 : 섬유 아세포는 높은 이동성이며 예컨대 상피, 내피 및 평활근 세포 등의 다른 종류의 세포에 비하여 증식. 가지 방법은 섬유 아세포의 이러한 기능을 이용하고, outgro 소요조직 섹션에서 날개 세포는 섬유 아 세포에 풍부하다. 여러 셀 통로 후, 다른 종류의 세포가 정제 된 섬유 아세포 세포 배양 결과, 생존 및 10 % 소 태아 혈청 (FBS)을 DMEM으로 전파하지 못한다. 초대 배양 세포는 다음 3-7 통로를 사용할 수있다. </li></ol></li><li> 세포 생장에 대한 절편 문화와 조화 : <ol><li> 수정 5 이전에보고 된 프로토콜을 사용하여 섬유 아세포의 차 문화를 수행합니다. </li><li> 실험실에있어서, 배양 배지가없는 10cm 조직 배양 접시에 조직 샘플을 배치하고 작은 섹션으로 잘라 ~ 멸균 겸자 메스를 사용하여 크기가 2 ~ 3 mm이다. 이 과정에서 다른 세포 생장의 효율이 손상 섹션 건조하게하지 마십시오. </li></ol></li><li> 상피 세포층과 결합 조직의 분리 : <ol><li> 4 ° C에서 16 시간 동안 배양 2,000 PU / ㎖ 디스 파제 I를 포함하는 매체와 문화에 절단 된 조직 절편을 만끽 해보세요. &lt;/ 리&gt; <li> 접시에 조직 절편을 전송하고, 인접한 결합 조직에서 상피 세포층을 분리합니다. 미생물의 오염을 방지하기 위해 클린 벤치의 섹션을 처리. 주 : 상피 세포의 초대 배양이 필요한 경우, 단계 130을 수행한다. 단 섬유 아세포가 고립 될 경우 후속 절편 배양 및 세포 통로 정제 된 섬유 아세포 인구를 산출 상피 세포에 대한 불리한 때문에, 1.3 단계를 생략합니다. </li></ol></li><li> 조직 절편의 첨부 : <ol><li> 두 메스를 사용하여 1mm 조각으로 조직을 말하다. 충분히 작은 조각이 아닌 경우에는 접시으로부터보다 쉽게​​ 분리하고, 세포는 접시 표면에 빨리 성장하지 못할 것이다. </li><li> 각 부분을 둘러싸는 빈 영역을 유지하기 위해 서로 떨어져 작은 폐 조직 섹션을 배치. 조직 절편의 부착을 용이하게하기 위해 메스 블레이드를 사용하여 조직 배양 접시의 표면에 흠집을 확인. 참고 : 긁는또한 세포 마이그레이션 할 수있는 함께 트랙을 제공, 세포 생장을 촉진하기 위해 나타납니다. <ol><li> 대안 적으로, 개별적 6- 웰 플레이트의 각 웰에 다진 조직편을 배치하고, 커버 슬립으로 그들을 커버. 실리콘 그리스를 사용하여 플레이트의 표면에 커버 슬립을 부착. </li><li> 각 웰 멸균 핀을 사용하여 1.5 cm 떨어진 두 지점에서 실리콘 그리스를 놓습니다. 조직편 커버리지 여러 통로 다음 세포 생장 및 세포 증식의 효과를 향상시킨다. </li></ol></li></ol></li><li> 이후 이식편 문화 : <ol><li> 샘플이 건조하면 효율적으로 세포 부산물은 대부분 손실로 조직 섹션은, 건조하기 전에 조심스럽게 접시에 배지를 추가합니다. 너무 빨리 조직 섹션은 분리하는 것처럼 매체를 붓지 마십시오. </li><li> 10 % FBS와 DMEM에서 배양 세포. 추가 부력에서 분리를 촉진대로, 부동 수 있다는 너무 많은 단지 조직을 충당하기에 충분한 매체를 사용하지만요리. </li></ol></li><li> 셀 통로 : <ol><li> 배양 접시의 반복​​ 운동이 조직 절편의 이탈로 이어질 수 있으므로 항상주의 문화 매체를 처리합니다. </li><li> 5-7일 37 ° C에서 인큐베이터에서 배양 접시를 놓습니다. 매일 매체를 새로 고침하고 매체 변경하는 동안 최소한의 배양 접시를 처리합니다. 섬유 아세포는 다음 몇 주 동안 조직 절편의 가장자리에서 자라다. 주 : ~ 2-3주 후 벗어나지 아세포 거의 표면적의 양에 따라, 합류에 도달한다. </li><li> 1X PBS로 세포를 씻으십시오, 그리고 나는 판에 트립신 1 ㎖를 추가합니다. </li><li> 트립신 처리 한 후, 10 % FBS가 보충 된 DMEM 9 ml의 세포를 사용하여 분리. 조직 절편과 함께 10 ㎖ 튜브 내의 매질에 현탁하고 세포를 모은다. 세포 펠렛을 형성하는 원심 분리 후, 새로운 배지에서 얻어진 펠릿을 재현 탁. </li><li> 새로운 막힌에 세포와 조직 섹션을 시드진짜야 요리, 어느 시점에서 조직 세포가 부착하고 성장하는 동안 요리에 연결하는 데 실패. 다음 날, 흡인에 의해 떠있는 조직 조각을 제거하고, 매체를 변경합니다. 첨부 섬유 아세포 이후 문화와 함께 전파. </li></ol></li></ol> 인간 폐 섬유 아세포 암세포 2. 입체 공동 문화 <ol><li> 세포 및 시약의 제조 : <ol><li> 약 2 × 10 5 상피 세포와 잘 당 2.5 × 10 5 섬유 아세포를 사용합니다. (3 ㎎ / ㎖, pH를 3), FBS (100 %), 재구성 완충액 (50 mM의 NaOH 등, 260 mM의 NaHCO3을 200 mM의 HEPES)가, 5X DMEM는, DMEM은 섬유 아세포 배양 용 10 % FBS가 보충 된 콜라겐 형 IA 준비 3D 및 공동 배양 배지 (1 : 암세포 섬유 아세포 배양 배지 및 배지 1 혼합물). </li><li> 문화 A549 폐 선암 세포, 섬유 아세포 및 DMEM에서 A549 차원 공동 배양은 10 % FBS로 보충. </li></ol></li><li> 콜라겐 겔 Formation : <ol><li> 1X PBS와 10cm 접시에서 배양 섬유 아세포를 씻고 난 판에 트립신 1 ㎖를 추가합니다. 37 ℃에서 ~ 5 분 동안 트립신 처리 후, 10 % FBS가 보충 된 9 ㎖의 DMEM을 사용하여 세포를 분리. 10 ㎖ 튜브에 세포를 배지를 수집하고, 세포 펠렛을 형성하도록 200-300 XG에이를 원심 분리기. </li><li> 5 × 105 / ml의 밀도로 100 % FBS에서 얻어진 펠렛을 재현 탁. </li><li> 얼음에 콜라겐 젤을 준비합니다. 다음 절차에 앞서 냉장고 시약 피펫 및 튜브 쿨. 심지어 얼음에, 혼합물을 어느 정도 고화하고, 총 부피는 모든 성분의 합보다 작다. </li><li> 각 웰에서, FBS에서 섬유 아세포 서스펜션 (2.5 × 10 5 세포)의 0.5 ml의 2.3 ml의 유형 IA 콜라겐, 670 ㎕의 5 배 DMEM, 330 ㎕를 재구성 버퍼를 혼합하여 콜라겐 젤을 준비합니다. 젤은 실온에서 쉽게 설정할 수 있습니다. <ol><li> 적극적으로 균질 마일을 보장하기 위해 피펫xture. 피펫 팅 과정에서 거품을 만들지 마십시오. </li></ol></li><li> 6- 웰 플레이트의 각 웰에 혼합물 (3 ㎖)를 첨가하고, 30-60 분 동안 방해없이 37 ℃ 배양기에서 허용 아교질로 만들다. </li></ol></li><li> 콜라겐 겔 암 세포 배양 : <ol><li> 6 수정을 이전에보고 된 프로토콜을 사용하여 입체 공동 문화를 수행합니다. </li><li> 1 × 105 / ㎖의 밀도로 3D 공동 - 배양 배지 (또는 A549 세포의 경우에는 10 % FBS를 가진 DMEM)에 재현 탁 암세포. </li><li> 각 겔의 표면에 재현 탁 된 세포 용액 (2 × 105 세포) 2 ㎖를 붓는다. 실험 조건에 따라 공동 배양 암 세포 또는 섬유 아세포의 세포 수를 수정한다. </li></ol></li><li> 젤의 수축 : <ol><li> 암 세포는 콜라겐 겔에 부착 할 수 있도록 37 ℃에서 밤새 인큐베이션 젤. 각 젤을 분리하고 O를 잘 각각 &#39;부동 문화&#39;를 생성6 잘 판 바. 주 : 부유 배양을 사용하여 겔 크기에 다양한 변경이 공 배양 암세포와 섬유 아세포 세포 간의 상호 작용에 의해 매개되는 기계적 장력 및 콜라겐 분해에 의해 발생된다. </li><li> 우물의 가장자리에서 각 젤을 분리 각도 21 G 바늘이나 작은 주걱을 사용합니다. 2-3 일마다이 3D 공동 배양 배지 2 ㎖로 웰을 새로. 5 일간 매일 각 겔의 크기를 측정한다. </li></ol></li><li> 콜라겐 젤의 분석 : <ol><li> 이러한 분석 Sircol 콜라겐 정량 방법을 사용하여 각각의 콜라겐 겔 함량을 측정. 젤 5에서 RNA를 수집 한 후 transcriptomic 분석 또는 정량 RT-PCR을 수행합니다. </li></ol></li></ol> 3. 공기 - 액체 인터페이스 문화 침략 분석 <ol><li> 오일, 37 ° C에서 콜라겐 젤을 배양 한 후, 새로운 6-에 메쉬 (70 ㎛의 공극 크기)을 배치하여 공기 수축 겔을 노출잘 접시. 유동 세포 계측법에 사용되는 시판 셀 스트레이너의 메쉬를 확인합니다. </li><li> 부드럽게 3D 공동 배양 배지 11 ml로 각각의 웰을 채운다. </li><li> 멸균 스푼을 사용하여 메쉬에 젤을 놓고 젤에 남아있는 모든 매체를 제거합니다. 공기가 겔의 상부 표면을 노출 동안이 상태에서, 중간 젤에서 잠수함. 공기 - 액체 인터페이스 등이 문화 조건을 정의합니다. 주 : 인큐베이터에서 37 ℃에서 공기 - 액체 계면을 유지 배양 5-7 일 후, 경부암 세포 겔로 이동한다. 세포 유형 및 셀룰러 상호 작용의 결과로서 따라, 암세포에서 세포의 형태 학적 변화의 가변 정도는 공기 - 액체 계면 배양에 의해 유도된다. </li><li> 조직 학적 평가를 위해 실온에서 밤새 포르말린 용액에서 겔을 고정하고 파라핀에 포함. 헤 마톡 실린 및 에오신 (H &amp; E)와 수직 섹션 (4 μm의) 얼룩​​. 면역 조직 화학 분석 O를 수행n은 섹션 4. </li></ol>

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Cancer. 6 (5), 392-401 (2006).</li><li>Calvo, 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=Mechanotransduction+and+YAP-dependent+matrix+remodelling+is+required+for+the+generation+and+maintenance+of+cancer-associated+fibroblasts.">Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts.</a> Nat. Cell Biol. 15 (6), 637-646 (2013).</li><li>Horie, M., 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=Histamine+induces+human+lung+fibroblast-mediated+collagen+gel+contraction+via+histamine+H1+receptor.">Histamine induces human lung fibroblast-mediated collagen gel contraction via histamine H1 receptor.</a> Exp. Lung Res. 40 (5), 222-236 (2014).</li></ol>

The authors declare that they have no competing financial interests.

CAFS 암세포 주변 ECM의 주요 구성 요소를 형성하고, 종양 만 골격을 제공 할뿐만 아니라 적극적으로 종양 발생에 관여하지 7. 축적 된 증거 CAF 매개 종양 진행 (8)의 중요한 역할을 강조 최종 예후에 CAFS 또는 관련 분자의 영향을 풀어 낸다. 이전 연구에서는 폐 CAFS (4)를 분리하는 가지 방법을 사용. 이 실험에서는, 접시 표면에 조직 절편 밀착성의 유지가...

Cancer tissue can be perceived as a type of organ, which evolves through close interactions between the cancer and the tumor stromal microenvironment, composed of cancer-associated fibroblasts (CAFs), immune cells, tumor vessels and the extracellular matrix (ECM). CAFs are the major source of soluble factors (cytokines, growth factors and chemokines) that exert mitogenic, pro-migratory and pro-invasive effects on cancer cells. They also stimulate tumor vessel formation and recruit precursor cells, such as bone marrow-derived cells (BMDC). Activated CAFs are involved in the production and remodeling of the ECM, thereby promoting the growth and spread of cancer cells1. CAFs also provide a niche that facilitates tumor cell colonization and metastasis and are capable of conferring stem cell phenotypes onto neighboring cancer cells. Pathological observations suggest that stromal reactions or fibrotic changes in cancer tissues are indicative of a poor prognosis. Recent studies have also demonstrated that tumor stromal features, such as the gene signature, can predict patient prognosis. Furthermore, CAF-derived factors can modulate sensitivity to chemotherapy, highlighting the role of CAFs in determining drug sensitivity and resistance2.
As CAFs play a multifaceted role in the promotion of tumor progression through signaling pathways that mediate interactions between CAFs and different cell types within the tumor microenvironment, they have attracted increasing attention as novel targets for cancer therapies. The heterogeneity of the cell populations within the cancer microenvironment presents an obstacle for targeting CAFs. Several markers for CAFs have been proposed, such as &#945;-smooth muscle actin (&#945;-SMA), fibroblast activation protein (FAP), and fibroblast specific proten-1 (FSP-1: also called S100A4); however, these molecular markers are not specific for distinguishing CAFs from other cells present in non-cancerous tissues3. Therefore, further studies are needed to obtain more knowledge about the specific properties of CAFs. To this end, it is informative to characterize primary cultured CAFs compared with patient-matched normal fibroblasts.
Recently, analyses on patient-derived CAFs have been reported in several cancer types, revealing unique gene expression patterns and cell behaviors compared with fibroblasts derived from non-cancerous tissues. Using isolated CAFs from human lung cancer tissues, we developed a three-dimensional co-culture method, enabling us to evaluate the properties of lung CAFs. In this model, we investigated the effects of the CAFs on lung cancer cell invasion, proliferation and collagen gel contraction, which experimentally recapitulated the tumor-promoting roles of lung CAFs4.

<table border="0" cellpadding="0" cellspacing="0" width="1035">
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			<td height="22" style="height:22px;width:267px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:471px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">DMEM</td>
			<td style="width:297px;">Sigma-Aldrich</td>
			<td style="width:471px;">D5796</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">FBS</td>
			<td style="width:297px;">GIBCO</td>
			<td style="width:471px;">10437</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Collagen type IA</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;">CELL-1A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Reconstitution buffer</td>
			<td style="width:297px;">Nitta gelatin Inc.</td>
			<td style="width:471px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cover slip</td>
			<td style="width:297px;">NUNC</td>
			<td style="width:471px;">174934</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Silicone grease</td>
			<td style="width:297px;">Dow Corning Toray</td>
			<td style="width:471px;">High vacuum grease</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Dispase I</td>
			<td style="width:297px;">WAKO</td>
			<td style="width:471px;">386-02271</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">6-well plate</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">353046</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Cell strainer (70 &#956;m)</td>
			<td style="width:297px;">BD Falcon</td>
			<td style="width:471px;">352350</td>
		</tr>
	</tbody>
</table>

three-dimensional co-culture model for tumor-stromal interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

이 공동 배양법, 종양 미세 환경을 흉내 낸 콜라겐 젤에 포함 된 암 세포와 섬유 아세포와의 상호 작용을 연구하기위한 유용한 도구이다. 콜라겐 겔 수축, 암세포의 침윤 및 형태 변화 : 이전의 연구에서, 세 개의 파라미터가이 실험 모델에서 평가 하였다. 암 세포의 증식은 또한 면역 염색 Ki67 4를 이용하여 추정 하였다. 폐 조직 샘플을 절제 폐 로브 (도 1A)의 암 및 암이 아닌 ?...

Watch this Scientific Journal Video about Three-dimensional Co-culture Model for Tumor-stromal Interaction at JoVE.com

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

종양 기질 상호 작용에 대한 세 가지 차원의 공동 문화 모델

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

three-dimensional-co-culture-model-for-tumor-stromal-interaction

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Medicine

17.2K 조회수.  The University of Tokyo. Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

비디오: Three-dimensional Co-culture Model for Tumor-stromal Interaction

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

인간 Mammary 섬유아 세포에서 암종 - 관련 섬유아 세포의 실험 세대 (CAFs)

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

연구

의학

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

인간 Neuroendocrine 종양 치료의 연구를위한 3 차원 모델로 인간 Neuroendocrine 종양 세포 라인

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

차 사람 골수 및 악성 종양을 연구하는 세 가지 차원 조직 배양 모델

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

3 차원 (3D) 모델을 사용하여 유방암 세포 침윤의 정량화

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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

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

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

인간의 뇌하수체 선종 세포의 3 차원 알긴산 비드 문화

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

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Myography 와이어를 사용 하 여 마우스 Mesenteric 동맥의 isometric 수축 측정

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

복잡 한 대동맥 변칙의 3 차원 인쇄

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The accuracy of PVP mainly depends on the surgeons&#39; experience and multiple fluoroscopes during a traditional procedure. Puncture related complications were reported all over the world. To make the surgical procedure more precise and decrease the rate of puncture-related complications, our team applied a three-dimensional printing guide template to PVP to modify the traditional procedure. This protocol introduces how to model target vertebrae DICOM imaging data into three-dimensions in the software, how to simulate operation in this 3-D model, and how to use all of the surgical data to reconstruct a patient specific template for application. Using this template, surgeons can identify suitable puncture points accurately to improve the accuracy of the operation. The whole protocol includes: 1) diagnosis of the osteoporotic vertebral compression fracture; 2) acquisition of CT imaging of the target vertebra; 3) simulation of the operation in the software; 4) design and fabrication of the 3-D printing guide template; and 5) application of the template into an operation procedure.

Three-Dimensional Printing Guide Template Assisted Percutaneous Vertebroplasty PVP

Herein, we present a three-dimensional printing guide template for percutaneous vertebraplasty. A patient with a T11 vertebral compression fracture was selected as a case study.

3차원 인쇄 가이드 템플릿 보조 경피적 척추 성형술 (PVP)

Percutaneous vertebroplasty (PVP) is considered an effective treatment for the back pain caused by osteoporotic vertebral compression fracture. The ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

Murine Hindlimb Gangrene 모델에서 발판 혈관 구조의 고해상도 입체 이미징

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...