3R01AR054458 - 02S2 :이 작품은 관절염과 Musculoskeletal 국립 연구소와 피부병에서 교부금에 의해 지원되었다.

1. 1 일 1) 플레이트 세포 <ol><li> 적어도 18-24시간 전에 라벨에 confluency 80 %에서 T75 플라스크에 플레이트 hMSC. 다른 선박에 대한지도를 위해 표 1을 참조하십시오. </li></ol> 2. 날 2 2) 라벨 솔루션을 준비합니다. 이 준비 400 μg 철 / ML의 농도와 confluency 80 % (1) T75 플라스크 레이블을 것입니다. 다른 선박에 대한지도를 위해 표 1을 참조하십시오. <ol><li> 15 ML 원뿔 튜브 : 혈청이없는 미디어 1 ML과 ferumoxytol의 93.1 μl (30 MG / ML 주식)를 혼합하여 솔루션 (해결책 1) 만듭니다. </li><li> 두 번째 15 ML 원뿔 튜브 : 혈청 무료 매체와 프로타민 황산 7 μl (10 MG / ML 재고 없음) 1 ML을 혼합하여 두 번째 솔루션 (해결책 2) 만듭니다. </li><li> 각 솔루션은 5 분 휴식을 허용합니다. </li><li> 함께 해결책 1과 2를 추가합니다. 부드럽게 새로운 라벨 솔루션을 섞는다. 5 분 휴식하는 솔루션 USPIO - PRO을 허용하도록 허용tamine 황산의 단지가 형성. </li><li> SPIO / 7 ML의 최종 볼륨 프로타민 황산 용액의 혼합이 ML에 혈청이없는 미디어의 5 ML을 추가합니다. </li></ol> 3) 라벨에 대한 전지를 준비 <ol><li> 세포에서 미디어를 기음. </li><li> 천천히 거리에 대비 에이전트 이해와 라벨 효율에 악영향을 수 잔류 혈청 단백질 및 기타 미디어 구성 요소를 씻어 사전 예열 MG / CA - 무료로 D - PBS 또는 혈청 프리 미디어로부터 2-3 ML로 한번 세포를 씻는다. 린스 액을 기음. </li></ol> 4) 레이블 셀 <ol><li> 세포로 해결책을 라벨의 전체 7 ML을 추가합니다. </li><li> 인큐베이터 (37 ° C / 5퍼센트 CO 2)와 세포를 4 시간 동안 상표 솔루션에 품어 수 있도록 장소에 세포. </li><li> 10 % 최종 혈청 농도를 달성하기 위해 세포의 라벨 솔루션으로 FCS 700 μl를 추가합니다. 혈청은 세포가 익숙한 어떤의 풍부한 환경을 다시 설정하고, MI에 도움에 추가됩니다돌연 변화의 혈청이없는 환경에 24 시간 노출 초래할 수 세포 죽음을 nimizing. </li><li> 세포가 추가로 20 시간 라벨 솔루션으로 품어 수 있습니다. </li></ol> 3. 3 일 5) 표시 전지를 준비 <ol><li> 세포의 라벨 솔루션을 기음. </li><li> 부드럽게 미리 예열 MG / CA - 무료로 D - PBS로부터 2-3 ML로 세포를 씻어. PBS를 기음. 그것은 MG와 CA는 다음 단계에서 트립신의 효율성에 악영향을 줄 것이다대로 MG / CA - 무료로 PBS를 사용하는 것이 중요합니다. </li><li> 세포에 미리 예열 0.05 %의 트립신 2 ML을 추가하고 술병의 전체 표면을 보장하기 위해 앞뒤로 술병을 기울이 트립신의 얇은 층으로 덮여있다. 인큐베이터 (37 ° C / 5퍼센트 CO 2)의 세포를 놓고 세포가 접시에서 분리 시작 때까지 세포 5~7분 또는 휴식을하실 수 있습니다. 그것은 현미경으로 부대를 확인 할 수 있습니다. 부드럽게 필요한 경우 FA로 술병의 측면을 탭세포 표면 부대를 cilitate. </li><li> 트립신을 중화하기 위해 술병에 사전 예열 전체 미디어의 4 ML을 추가합니다. 부드럽게 여러 번 트립신 / 미디어 / 셀을 솔루션을 pipetting 아래로 술병을 씻어. 15 ML 원뿔 튜브에 전체 솔루션을 수집합니다. 5 분 400 RCF의 셀 솔루션을 원심 분리기. </li><li> 세포 펠렛을 방해하지 않고 뜨는를 주의깊게 기음. 미디어 5 ML (혈청 포함하거나 혈청 무료)에있는 세포를 Resuspend. 5 분 400 RCF의 셀 솔루션을 원심 분리기. </li><li> 단계 5.5에서 설명한 세포 세척을 반복합니다. 전체에서 세포는 미디어와 세포의 표면에 잔류, 무료 대비 에이전트를 제거하는 세 번 씻어서 것입니다. </li><li> 세포가 이전 세척시 손실됩니다로서 가장 정확한 셀 카운트를 달성하기 위해이 시점에서 세포를 세어보세요. 세포의 생존 테스트를 수행합니다. 세포는 이제 이후 분석 및 실험 또는 응용 프로그램에 대한 준비가되어 있습니다. MR 영상은과 T1 - W로 수행할 수<html> T2 - W 매개 변수, ferumoxytol는 T2 - W 대비 에이전트이지만 때문에 ferumoxytol는 T1 효과를 전시하고 있습니다. 이미지 ferumoxytol 표시 전지하는 데 사용할 수있는 대표 T2 - W MRI 매개 변수는 다음과 같습니다 : 60-80 MS의 2000년부터 2500년까지 MS와 TE의 TR와 FSE 또는 SE_Multislice 시퀀스를. T1 - W 이미지를 얻기위한, FSE에서 TE 값 또는 SE_Multislice 시퀀스를 감소하거나 높은 TR과 낮은 TE와 GRE 시퀀스를 사용합니다. 그림 4는 표시 줄기 세포 이미징에 대한 생체내 응용 프로그램에서 가능성을 보여줍니다. </li></ol> 4. 대표 결과 : 라벨 세포 T2 - 가중 MR 이미지에서 어둡게 또는 중대한 부정적인 대비 효과 및 T1 - 가중 MR 이미지에 이트닝이나 긍정적인 대비 효과 (그림 2)를 보여줍니다. 세로 MR 이미징 및 생존 능력 테스트는 오일 게시물 라벨이 더 큰 MR 신호 및 레이블이 지정되지 않은 컨트롤 (데이터가 표시되지 않음)에 비해 생존 능력에없이 큰 영향을 증명하지 수행. jove_content &quot;&gt; 라벨 줄기 세포는 이후 다양한 세포 유형으로 차별하거나 정맥 생체내 조사에 대한 주입 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3482/3482fig1.jpg" /> 그림 1. 철 산화물 nanoparticle, ferumoxytol와 함께 줄기 세포를 라벨에 대한 도식 시간 라인. <img alt="그림 2" src="/files/ftp_upload/3482/3482fig2.jpg" /> 그림 2. 세포 펠렛의 세포와 화살 크로스 섹션 thrrough eppendorf 튜브의 MR 이미지. A) 컨트롤 레이블이 지정되지 않은. B) 상당한 T2 또는 부정적인 대비 에이전트 효과 T2 - 가중 FSE 또는 SE_Multislice 영상과 T1 - 가중 GRE 시퀀스에서 T1 또는 긍정적인 대비 에이전트 효과를 입증 라벨 세포. <img alt="그림 3" src="/files/ftp_upload/3482/3482fig3.jpg" /> 그림 3. 셀 화소의 세포와 eppendorf 튜브의 화살 크로스 섹션수 있습니다. 조금은 10,000으로 표시 ferumoxytol 세포 T2 - W MR 영상을 통해 검색하실 수 있습니다. <img alt="그림 4" src="/files/ftp_upload/3482/3482fig4.jpg" /> 그림 4. ferumoxytol - 표시 줄기 세포의 MR 시각화는 murine 대뇌 심실로 주입. USPIO - 표시 줄기 세포없이 주사로 murine barin의) 축방향, MR 이미지. B) 코로나 축방향, C) 및 D) 화살 MR 이미지는 murine 머리에 주입 USPIO - 표시 줄기 세포 (흰색 화살표)의 T2, 부정적인 대비 효과를 묘사. <img alt="표 1" src="/files/ftp_upload/3482/3482table1.jpg" /> 다른 혈관에서 세포를 라벨에 대한 표 1. 수량 adaptions.

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이 연구에 대한 모든 참여자는 공개를 제공하지 않습니다.

줄기 세포 engraftments의 효능을 개선하는 것은 재생 의료의 발전을 위해 중요합니다. 생체내의 줄기 세포에 대한 비침습 시각화 기술은 크게 성공 engraftment의 결과로 이어질 메커니즘을 이해하는 능력을 향상시킵니다. 이러한 우리가 보여준 프로 시저와 같은 MR 시각화, 대한 자기 상표는 MR 영상과 함께 줄기 세포의 생체내 추적하실 수 있습니다. 자기 (磁气) 표시 줄기 세포는 이전 대?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> D - 가상 높은 포도당 </td><td> 시그마 </td><td> D5648 </td><td> 사용할 수 원하는 줄기 세포 라인을위한 또는 다른 기본 매체 </td></tr><tr><td> D - PBS (CA + +, MG + + 무료) </td><td> GIBCO </td><td> 14190-144 </td><td></td></tr><tr><td> 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 0.05 % </td><td> Invitrogen </td><td> 25300-120 </td><td></td></tr><tr><td> 태아 소 혈청 (FBS) </td><td> Hyclone </td><td> SH30071.03 </td><td></td></tr><tr><td> Ferumoxytol (Feraheme) </td><td> AMAG </td><td> 59338-0775-​​01 </td><td></td></tr><tr><td> 프로타민 황산 </td><td> APP 알약. </td><td> 22,930 </td><td></td></tr>
</tbody>
</table>

labeling stem cells with ferumoxytol, an fda-approved iron oxide nanoparticle

줄기 세포 기반 요법은 재생 의료 분야에 대한 상당한 잠재력을 제공합니다. 그러나, 많은 이식 세포의 생체내 반응 속도론에 관한 이해에 남아 있습니다. 반복 생체내에 이식 줄기 세포를 모니터하는 비침습 방법은 수사관이 직접 줄기 세포 이식을 모니터링하도록하고 성공 여부 engraftment 결과를 확인합니다. 줄기 세포의 다양한 수많은 어플 리케이션을위한 조사하고 있습니다. 인간의 배아 신장 293 (HEK293) 세포, 인간 mesenchymal 줄기 세포 (hMSC)과 유도 pluripotent 줄기 (IPS) 세포 :이 프로토콜은 3 개의 줄기 세포 집단에 초점을 맞추고 있습니다. HEK 293 세포는 가지각색으로 아데노 바이러스 5 DNA와 문화 속에서 자란 인간의 배아 신장 세포에서 파생됩니다. 그들이 쉽게 양식 때문에 이러한 세포가 광범위하게 연구에 사용되고, 빠르게 성장하고 쉽게 transfected 수 있습니다. hMSCs는 성인 골수에서 발견됩니다. 이러한 세포 CAmultipotency 또는 세포 운명의 제한된 수의으로 차별화할 가능성을 유지하면서 N은 undifferentiated 세포로 복제됩니다. hMSCs는 osteoblasts, adipocytes, chondrocytes, 힘줄, 근육 및 골수의 기질을 포함 mesenchymal 조직의 lineages로 구별하실 수 있습니다. IPS 세포는 유전자 유전자와 배아 줄기 세포의 정의 속성과 비슷한 요소를 표현하기 위해 수정되었습니다 성인 세포를 다시 프로그램입니다. 이러한 세포들은 모든 세포 lineages 1로 차별화하는 능력을 가지고 pluripotent 의미입니다. 모두 hMSCs와 IPS 세포는 조직 재생 능력 - 생체내를 증명하고있다. 함께 superparamagnetic 철 산화물 (SPIO) nanoparticle 셀 라벨의 사용과 자기 공명 (MR) 영상은 가까운 미세한 해부 해상도에 의한 줄기 세포의 생체내 추적에 효과 입증, 그 이상 혈액 반감기는 세로 이미징과를 허용 높은 sensitivitSPIO의 MR 이미징에서 제공하는 세포 감지 y는 2-4 nanoparticles. 또한, SPIOs의 사용과 MR 영상은 임상적으로 번역입니다. SPIOs는 플라즈마 구성 요소에서 bioreactive 철 코어를 포함하는 역할을 dextran, carboxydextran 또는 전분 표면 코팅과 철 산화물 코어로 구성되어 있습니다. 이러한 에이전트는 T2 - 가중 MR 이미지 5 감소 신호로 이어질 현지 자기장 inhomogeneities을 만듭니다. 불행하게도, SPIOs은 더 이상 생산되고 있지 않습니다. 2 세대, ultrasmall SPIOs (USPIO)는, 그러나, 대안을 제공합니다. Ferumoxytol (FerahemeTM)는 polyglucose sorbitol carboxymethylether의 코트로 둘러싸여 아닌 stoichiometric 자철광 코어로 구성된 하나 USPIO입니다. ferumoxytol의 콜로이드, 입자 크기는 빛의 산란에 의해 결정 17-30 NM됩니다. 분자량은 750 kDa이며, 2T MRI 분야에서 상수 relaxivity은 58.609 MM - 1 - 1 초 강도 4. Ferumoxytol은 최근 FDA 승인되었습니다신장 장애 환자 6 철분 결핍증의 치료에 S는 철 보충. 우리 그룹은 셀 라벨 응용 프로그램을위한 &#39;오프 라벨&#39;사용이 에이전트를 적용했습니다. 우리의 기술은 MR 이미지에 라벨 세포의 중요한 MR 신호 효과를 리드 ferumoxytol와 줄기 세포의 효율적인 라벨을 보여줍니다. 이 기술은 사전 임상 및 임상 설정에서 줄기 세포 요법의 비침습 모니터링 적용될 수 있습니다.

Watch this Scientific Journal Video about Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle at JoVE.com

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

우리는 FDA 승인, superparamagnetic 철 산화물 (SPIO), ferumoxytol (Feraheme)와 줄기 세포를 라벨링 및 추적을위한 기술을 설명합니다. 시각화에 대한 자기 공명 (MR) 영상을 이용하여이 세포 이미징 기술은 환자의 성공 여부에 줄기 세포 engraftments의 장기 모니터링 및 진단을 위해 쉽게 액세스할 수 있습니다.

Ferumoxytol, FDA 승인 철 산화물 Nanoparticle과 함께 줄기 세포를 라벨링

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

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20.7K 조회수.  Molecular Imaging Program at Stanford (MIPS). 우리는 FDA 승인, superparamagnetic 철 산화물 (SPIO), ferumoxytol (Feraheme)와 줄기 세포를 라벨링 및 추적을위한 기술을 설명합니다. 시각화에 대한 자기 공명 (MR) 영상을 이용하여이 세포 이미징 기술은 환자의 성공 여부에 줄기 세포 engraftments의 장기 모니터링 및 진단을 위해 쉽게 액세스할 수 있습니다.

비디오: Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

인간 치과 펄프의 줄기 세포의 분리, 특성화 및 비교 차별화 두 가지 방법을 사용하여 영구 치아에서 파생

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

연구

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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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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 ...

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. Repeated transfusions of packed red blood cells (PRBCs) is currently the simplest and most used technique for chronic transfusion programs. However, iron overload is one of the major side effects of this therapy. More developed methods exist, notably the apheresis of RBC (erythrapheresis), which is currently the safest and most efficient method. However, it is costly, complicated, and cannot be implemented everywhere, nor is it suitable for all patients. Manual exchange transfusions combine one or more manual phlebotomies with a PRBC transfusion.
At the Reference Center of Sickle Cell Disease, we set up a continuous method of manual exchange transfusion that is feasible for all hospital settings, demands no specific equipment, and is widely applicable. In terms of HbS decrease, stroke prevention, and iron overload prevention, this method showed comparable efficiency to erythrapheresis. In cases where erythrapheresis is not available, this method can be a good alternative for patients and care centers.

We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

겸상 적혈구 질환을 가진 환자에 대한 지속적인 수동 교환 수혈 : 철 과부하를 방지 할 수있는 효율적인 방법

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

초초파라마그네틱 산화질소 나노프로브의 합성, 특성화 및 적용

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Polyethyleneimine 코팅 된 산화 철 나노 입자 작은 간섭 RNA의 세포에 배달 하는 수단으로 생체 외에서 그리고 Vivo에서

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

인간 지방분지 유래 중간엽 줄기 세포의 분리 및 확장을 위한 효소 없는 방법

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

외상성 뇌 손상 뮤린 모델에서 비강 내 전달 후 MRI를 사용하여 수퍼 파라마그네틱 철 산화물 표지 메젠키말 줄기 세포 추적

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

칼슘 플루오로포어 라벨링 및 나노입자 추적 분석을 사용하여 인간에서 오줌 나노 결정의 추정

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...