이 연구는 플로리다의 BANKHEAD - 콜리 팀 과학 그랜트 (2BT03), 건강 / 국립 암 연구소 (1R21CA164322-01) 및 모 피트 암 센터의 팀 과학 부여의 국립 연구소의 국가가 투자되었다. 이 작품은 H. 리 모 피트 암 센터 연구원, NCI 지정 종합 암 센터 (P30-CA076292)에서 중개 연구 핵심 시설에 의해 부분적으로 지원하고있다. 일차 전지에 대한 액세스는 모 피트 암 센터에서 전체 암 관리 프로토콜을 통해 가능하게되었다.

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+bone+marrow+plasma+cell+infiltrates+in+multiple+myeloma:+the+added+value+of+CD138+immunohistochemistry.">Assessment of bone marrow plasma cell infiltrates in multiple myeloma: the added value of CD138 immunohistochemistry.</a> Hum Pathol. 38, 1779-1787 (2007).</li><li>Najar, 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=Mesenchymal+stromal+cells+promote+or+suppress+the+proliferation+of+T+lymphocytes+from+cord+blood+and+peripheral+blood:+the+importance+of+low+cell+ratio+and+role+of+interleukin-6.">Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6.</a> Cytotherapy. 11, 570-583 (2009).</li><li>Scott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+i+trialist.">Phase i trialist.</a> Lancet Oncol. 13, 236 (2012).</li></ol>

The authors have no conflict of interest to disclose.

요약하면, 이것은 골수 재건 차 MM 세포 및 약물의 생체 약력학의 감수성을 정량화하는 강력하고 높은 처리량 방법이다. 이 프로토콜 내에서 중요한 단계는 우물의 적절한 파종하고 적절한 (예상 결과에 대한 그림 2 참조) 초점 : MM과 기질 세포가 균일하게 분포되어 있는지 확인, 그 기질 세포는 우물의 바닥에 부착되어 MM 모든 세포는 MM 세포 어두운 링으로 둘러싸인 밝은 디스?...

이 방법의 목적은 이들 결과 chemoresistant 서브 식별 할 수있는 충분한 정밀도로, 생리 학적 조건에 가능한 한 가깝게, 생체 에이전트 패널 일차 전지를 다발성 골수종 (MM)의 약물 감수성을 특성화하는 것이다 종양 부담하고, 집단 내의 궁극적 임상 응답을 추정하기위한 계산 모델 파라미터 화. 전산 모델은 암 호스트 치료의 상호 작용과 같은 복잡한 시스템을 분석 할 수있는 강력한 도구입니다. 그러나 모델은 데이터를 매개 변수화하는 데 사용되는만큼 좋다. 불행하게도, 문헌에서 사용할 수있는 대부분의 데이터는 종종 상호 배타적 실험 조건에서 수득 된 바와 같은 모델을 파라미터 화하는 현재의 형태로 사용될 수 없다. 따라서, 새로운 실험은 종종 필요한 실험 파라미터 세트를 얻는 것을 목표로해야한다. 대부분의 생존 분석은, 그러나, 파괴적이고 일이다우리는 시간 지점 소수 종종 단지 하나에 제한. 이것은 크게 이러한 실험은 시스템의 시간적 동역학에 관한 정보를 제공하지 못한다 감수성 때문에 분석의 사용은, 계산 모델을 매개 변수화 장애. 이것은 종종 따라서 가능한 실험 조건의 수를 제한하는, 샘플 당 몇 백만에 한정 생검 후 짧은 수명을 갖는다 일차 암세포, 특히 사실이다. 또한 대부분의 생존력 분석법은 상기 세포를 교란하고 행할 수 실험의 수를 제한하는, 프로토콜의 추가 작업을 추가 정량의 시간에 비 - 암 (기질) 세포에서 암의 분리를 필요 . 전에 40 년, 연어와 공동 작업자 1은 종양 세포의 감수성의 평가에 대한 시험 관내 콜로니 형성 분석에서 그 유명을 제안했다. 불행하게도,이 분석은 여러 MYE 인해 소수로 혼합 성공을제어 조건에서 콜로니를 형성 할 수 있었다 로마 (MM) 환자 샘플 : 그것은, MM 세포의 0.1 %로 0.001 %의 작은 서브 그룹은 복제 할 수 있다고 추정은 다발성 골수종 줄기 세포를 2로 지칭된다. 이로 인해, 심지어 더 최근의 모델 3에서, 분석의 성공률과 동시에 시험 할 수있는 약물의 수를 제한. (표준 또는 실험) MM 에이전트 사 년 전보다 더 많은 경우이 문제가 훨씬 더 중요하다. 두 환자는 특정 약물에 대한 &quot;민감한&quot;또는 &quot;내성&quot;이다 : 이러한 초기 분석의 주요 한계는 이분법 출력된다. 어떤 정보는 종양 인구의 감도와 이질성의 정도에 대한 제공되지 않습니다. 따라서, 빠르게 성장하는 내성 세포의 작은 하위 집단 환자가 치료에 &quot;민감한&quot;로 분류 될 수 있지만, 곧 재발 것입니다, 따라서하지 벤치료의 혜택을 받고 있습니다. 암 환자의 생존율 4,5- (OS)에서의 응답 시간의 중요성을 감안할 때, 이러한 분석이 올바르게 일관 OS를 추정 할 수 없었다 얼마나 명확하다. 순서 이러한 한계를 회피하고, 약물의 패널 맞춤 임상 응답을 추정 할 환자 별 계산 모델을 생성 할 수 있도록, 우리는 다발성 골수종 (MM) 세포 라인의 비파괴 시험 약물 민감성 방법을 개발 및 세포 외 기질과 기질 6 포함한 골수 미세 환경의 생체 재건 일차 MM 세포. 이 분석은, 그러나, 그 크기 및 비용 장비의 특정 부분에서 한 번에 수행 될 수있는 실험이나 약물의 수를 제한 특히 상업용 마이크로 유체 슬라이드에 의존하는 한계가 있었다. 여기에 설명 된 시스템은 높은으로이 원래의 분석을 확장비파괴 세포 생존율을 정량화하는 디지털 이미지 분석 알고리즘에 기초하여 약물의 시험 관내 스크리닝의 Organotypic 용량 반응 플랫폼, -throughput. 도 384 또는 1536 웰 플레이트의 각 차 MM 세포 외 기질 및 간질 환자 유래 성장 인자를 포함하여 골수 미세 환경의 3D 재구성이다. 라이브 현미경과 디지털 영상 분석은 용량 - 반응 표면을 생성하는 데 사용되는 다른 약물 농도에서 세포 사멸 이벤트를 검출하기 위해 사용된다. 시험 관내 데이터로부터 수학적 모델은 환자의 종양 부담 내의 크기 및 하위 집단의 감수성을 식별하고, 종양 (임상 처방 6에서 생리 학적 조건에 약물 (들)에 반응하는 방법을 시뮬레이션하기 위해 사용될 수있다 그림 1). 이 플랫폼의 주요 혁신은 다음과 같습니다 약물 승낙 당 필요한 암 세포의 () 작은 수 (1,000 ~ 10,000배급); (b) 약물 효능의 생리적 조건 (세포 외 매트릭스, 간질 환자 유래 성장 인자)의 평가; (c) 만 명 시야 촬상 보낸 생존 마커 (7)로부터는 독성, 생물 발광 형광 8 또는 9 세포를 형질 감염시키는 것이 필요도 사용되지 않는다; (d)는 연속 화상 농도 및 노출 시간 (약물 동력학)의 함수로서 약물의 효과를 제공한다; 및 (e) 시험 관내 및 전산 진화 모델과의 통합, 임상 적 결과 (6) (실바 외., 준비)를 추정한다. 디지털 이미지 분석 알고리즘이 간질 세포에서 암을 분별할 수 있도록 핵심 요소 중 바닥에 서로 다른 친 화성이다 웰 : MM 세포 부착, 및 간질 세포의 바닥에 부착하면서, 매트릭스에 현탁 유지하지 잘하고 스트레칭. 이러한 분리는 MM 및 심지어 기질에도 발생동시에 접종 및 배양 O / N 프로세스 중에 발생 재. 플레이트의 원심 분리기에서 스핀 다운 시딩 다음은이 과정을 더욱 가속. 기질이 낮은 프로파일과 어두운 그늘 (그림 2)을 유지하면서 결과적으로, MM 세포는 어두운 링으로 둘러싸인 원형 밝은 디스크와 같은 이미지에 나타납니다. 알고리즘의 조정 등의 크기 및 형상과 같은 다른 형태 적 특징에 기초하여 별도의 암과 기질로 할 수 있지만 이와 같이, 현재의 형태에서의 분석은, 비 - 부착 암세포에 가장 적합하다. 이 프로토콜에서 우리는 (골수 유래 중간 엽 줄기 세포) BMSC와 HUVEC를, 골수 간질 및 혈관 주위의 틈새를 대표로, 세포 외 기질 (콜라겐 및 기저막 매트릭스)의 두 종류뿐만 아니라 공동 배양 두 가지 유형을 서술 각각. 384- 웰 플레이트를 사용하여 약물 농도 및 당 5 개의 복제, 우리는 TES 수 있었다단일 환자의 샘플 (31) 다른 약물에 T 최대. 기업도 1은 로봇을 이용한 피펫 최적 분포를 나타낸다 1536 웰 플레이트에서이 수는, 3은 수동 세포 시딩 사용되는 레이아웃 (127)을 도시도이다. 그림 3에서 디자인, 각 384 웰 플레이트 (21) 약물 테스트 (7) 다른 약물, 또는 한 환자 샘플 3 환자 샘플을 수행 할 수 있습니다. 각각의 약물 농도는 5로 표시되고, 각각의 상태는 중복 접종된다. 4 제어 우물은 (어떤 약물이 추가되지 않습니다) (7) 약물 (예, 우물 36-39, 126-129 및 200-203)의 각 세트에 대한 씨앗을 품고있다. 사용되는 약물의 효능을 확인하기 위해 인간 골수종 세포주 (예, H929 또는 MM1.S)도 시딩하고 (웰 76-89)를 사용 약제의 각각의 가장 높은 농도에서 중복 마취이다. 양 세포주 제어는 투여 량 반응 곡선을 비교 AC 때문에 사용 약물, 충분한 효능을 가질 수 있습니다로스 실험. 두 개의 웰 (75 웰, 90) 촬상시 벤치 탑 인큐베이터에서 발생할 수있는 문제를 검출하는, 즉, 환경 조건에 대한 제어와 같은 골수종 세포주로 접종된다. 웰 열거 획득 시간을 감소시키고, 장비의 마모를 감소 현미경 동력 스테이지에 의해 덮여 거리를 감소시키기 위하여 지그재그 패턴을 따른다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EVOS FL AUTO</td>
			<td>AMG</td>
			<td>AMAFD1000 + AMC1000</td>
			<td>Digital microscope equipped with motorized stage and bench top incubator.</td>
		</tr>
		<tr>
			<td>384-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3683 SIGMA</td>
			<td>Corning CellBIND 384 well plates 
			384 well plate, polystyrene, CellBIND surface, sterile, clear flat bottom, black, w/lid</td>
		</tr>
		<tr>
			<td>1,536-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3832 ALDRICH</td>
			<td>Corning 1536 well plates, low base, surface treatment CellBIND, sterile</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-1440-02 SIGMA</td>
			<td>For in vitro isolation of lymphocytes from human peripheral blood.</td>
		</tr>
		<tr>
			<td>CD138 magnetic beads</td>
			<td>Miltenyi&#160;</td>
			<td>130-051-301</td>
			<td>Antibody conjugated magnetic beads for selection of CD138+ cells.</td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi&#160;</td>
			<td>&#160;130-042-401</td>
			<td>Column for separation of cells.</td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Miltenyi&#160;</td>
			<td>130-042-302</td>
			<td>Magnet for separation column.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E6909 SIGMA</td>
			<td>ECM Gel from Engelbreth-Holm-Swarm murine sarcoma</td>
		</tr>
		<tr>
			<td>Getrex</td>
			<td>Life Technologies</td>
			<td>A1413201</td>
			<td>LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>Life Technologies</td>
			<td>C-003-25P-B</td>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC )</td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>GIBCO</td>
			<td>11875-093</td>
			<td>RPMI 1640 Medium + L-Glutamine + Phenol Red</td>
		</tr>
		<tr>
			<td>FBS Heat inactivated</td>
			<td>GIBCO</td>
			<td>10082-147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>3.1mg/mL Bovine collagen type I</td>
			<td>Advanced Biomatrix</td>
			<td>5005-B</td>
			<td>Bovine Collagen Solution, Type I, 3 mg/ml, 100 ml</td>
		</tr>
		<tr>
			<td>Precision XS</td>
			<td>BIOTEK</td>
			<td>PRC3841</td>
			<td>Robotic pipettor system</td>
		</tr>
	</tbody>
</table>

an organotypic high throughput system for characterization of drug sensitivity of primary multiple myeloma cells

본 연구에서 우리는 환자 유래 다발성 골수종 (MM) 세포의 감수성 및 이질성을 추정 생체 약물 민감성 분석 및 디지털 이미지 분석을 결합한 새로운 접근법을 설명한다. 이 방법은 환자 - 갓 외 기질 (콜라겐 또는 기저막 매트릭스)과 간질 (각각 포함하는 골수 미세 환경의 재구성 구성된 멀티 웰 플레이트에서 구현 미세 유체 챔버 내로 골수 흡 인물에서 추출 일차 MM 세포를 시드로 이루어져 유래 중간 엽 줄기 세포) 또는 인간 유래 혈관 내피 세포 (HUVEC를). 챔버는 다른 에이전트 및 농도로 마취하고, 디지털 카메라가 장착 된 전동식 현미경, 명 시야 현미경을 통해 96 시간 동안 순차적으로 촬상된다. 디지털 영상 분석 소프트웨어가 존재 또는 멤브레인의 움직임의 유무를 라이브 및 사균을 검출하고, 함수 O로 생존의 변화 곡선을 생성F 약물 농도와 노출 시간. 우리가 각각의 약물에 대한 종양의 감수성 인구의 파라미터뿐만 아니라 종양 이종 척도로서 본 하위 집단의 수를 결정하는 연산 모델을 사용한다. 이러한 환자 맞춤형 모델은 치료법을 시뮬레이트 및 임상 응답을 추정하는데 사용될 수있다.

실험의 흐름은 간단히도 1에서 설명하는 모든 단계가 성공적으로 완료되는 경우, 현미경에 의해 얻어지는 화상이도 2와 동등해야한다 :. MM 세포 명확 밝은 디스크 및 BMSCs 또는 HUVEC를 거의 볼 수 있어야 보지되어야 라이브 잘 배경 분포. 도 2a에 도시 된 바와 같이, MM 세포는 한 환자로부터 다른 크기가 다양하지만, 일반적으로 그들은 세포주보다 작다. 그들이 ...

Watch this Scientific Journal Video about An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells at JoVE.com

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

차 다발성 골수종 세포의 약물 민감도의 특성에 대한의 Organotypic 높은 처리량 시스템

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

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8.5K 조회수.  H. Lee Moffitt Cancer Center and Research Institute. We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

비디오: An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

뇌의 Organotypic 슬라이스와 암 세포의 3 차원 회전 타원체와 공 배양 시스템

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

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High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

신경 보호 전략에 대한 치료 요인의 납품을위한 설계 성인 줄기 세포의 높은 처리량 특성

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

닭에서 인간의 다발성 골수종 이종 이식 모델의 설립은 종양의 성장, 침략과 혈관 신생을 공부하기

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

약물 치료를 종양 미세 환경을 공부하고 대상에 대한 뇌종양 / Organotypic 슬라이스 공동 문화 시스템

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Ivacaftor, 그것의 주요 대사 산물, 및 생물 학적 체액의 낭 성 섬유 증 환자에서 Lumacaftor의 임상 샘플의 높은 처리량 분석에 대 한 최적화 된 LC-MS/MS 방법

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

생체 외에서 혈액-뇌 장벽 돼지 뇌 내 피 세포를 기반으로 하는 모델의 설립에 대 한 방법 개선

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

기간 Placentae의 태아 세포 막에서 기본 인간 Decidual 세포의 고립

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

고처리량 전기화학발광 7-Plex 분석으로 제1형 당뇨병 및 다발성 자가면역질환 동시 스크리닝

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

소설 인간 오르가노피 망막 문화 기술의 특성화

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...