우리가이 일을하는 동안 그들의 친절 지원하고 유익한 조언을위한 RIKEN - BSI 우리의 동료 주셔서 감사합니다. 교수님 시게미 모리 교수 G 보르 Juh SZ, DRS에게 진심으로 감사드립니다. 내가 고급 연구 대학원 대학, 일본에서 대학원 과정 중에 microdialysis 방법을 공부하는 것이 좋다 Kiyoji 마쓰야마, 카탈린 AK kesi 및 RP D Dobolyi. 마지막으로, 우리는 비디오 녹화에서 지원 씨 크리스 Sarnecky 박사 Akitsugu 다카스 감사합니다.

1 단계. 프로브 주입의 준비 <ol><li> 마취 (비디오에 표시되지 않음) interperitoneal 300ml의 주사 (12.5 밀리그램 / kg 체중)와 외과 마취의 안정 수준을 생산 xylazine (25 MG / kg) 혼합 각 새 마취. </li><li> stereotaxic 프레임에 머리를 수정 귀 바 &#39;압력의 로컬 통증을 줄이기 위해 Xylocaine (TM) 젤리를 사용합니다. 조류의 머리는 귀 바, 45 °의 각도로 머리를 가지고 부리 홀더 구성된 stereotaxic 장치에서 해결되었습니다. 이식에 대한 stereotaxic 수술하는 동안, 열 제어 외과 패드에 의해 조류의 시신을 따뜻하게 (온도 = 34 ~ 36 ° C). </li><li> 외과 영역을 소독 수술하기 전에 isodine (TM)으로 수술 부위를 소독. </li></ol> 2 단계. Stereotaxic 위치 <ol><li> 기준점 정렬 람다 (Y - 포인트) 마크 Y - 포인트 염료 채워진 피펫과 함께하는 더미 프로브 (유리 micropipette)를 맞춥니다. 마지막 추적 연구 남은 Dextran - TMR은 비디오에 사용되었습니다. </li><li> 피부의 절개 피부를 제거하고 두개골의 표면을 쉽게받을 수 있습니다. 그런 다음, 면화 면봉으로 두개골의 표면을 청소하십시오. </li><li> 가짜 정맥의 위치 (수평 방향) 대상 사이트 (AP 축과 래터럴 조정)에 더미 정맥으로 이동 </li><li> 소형 craniotomy 및 두라 메이터의 절개. craniotomy에 대한 좋은 포셉 및 두라 메이터의 incisions에 대한 훌륭한 주사 바늘 (30 G)를 사용합니다. 그런 다음, 두개골의 표면을 청소하십시오. </li><li> 가짜 정맥의 최종 위치 (주입) microdialysis 프로브 - 목표는 지역 위 0.30 mm에서 가이드 정맥의 끝부분을 찾습니다. 깨끗하고 건조한 표면이 거친 결합이 필요하기 때문에 마지막으로, 다시 두개골의 표면을 청소하십시오. </li></ol> 3 단계. 두개골 표면 전처리 (치과 제품의 &quot;직접 작성 복원&quot;에 대한 유사한 절차가 등장한다.) <ol><li> 프라이머 처리 <ol><li> 접시에 A와 B를 혼합 PRIMAR 액체. </li><li> 작은 브러시와 함께 두개골의 표면에 혼합물을 적용합니다. </li><li> 30 초 동안 둡니다. </li><li> 부드러운 공기 흐름과 건조. (세척하지 마십시오.) </li></ol></li><li> 접착제의 응용 <ol><li> 작은 브러시와 함께 두개골의 표면에 접착제를 적용합니다. </li><li> 부드러운 공기 흐름을 적용합니다. (세척하지 마십시오.) </li><li> 20 초 동안 빛을 치료. </li></ol></li></ol> 4 단계. 합성 수지 장착 <ol><li> 일반 충전기로 마운트 수지 일반 충전기를 사용할 수없는 경우, 마이크로 주걱 대신 사용할 수 있습니다. </li><li> 라이트 치료 </li><li> 증분 설치 증분 설치는 수지의 완전한 반응을 보장하는 것이 좋습니다. 증분 설치를위한 모든 설치하기 전에 접착제를 적용합니다. </li><li> 최종 경화에 대한 합성 수지 및 가벼운 치료를 몰드. </li><li> 수술의 끝 </li></ol> 외과 영역을 청소 (Gentamicin의 연고) 항생제 적용하고, stereotaxic 불꽃에서 동물을 제거합니다. 그런 다음, 온도 제어 챔버 (온도 = 33-35 ° C) 후 마취 회복을 위해 동물을 유지. 마찬가지로 비디오와 같이, 더 이상 문제는 수술 후 다음날에있는 동물의에서 관찰되지 않았습니다.

<ol>
	<li>Adell, A., Artigas, F., Boulton, A., Baker, G., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Brain+microdialysis:+Principles+and+applications.">In vivo Brain microdialysis: Principles and applications.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Kendrick, K., Robinson, T., Justice, J., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+in+large+unrestrained+animals:+neuroendocrine+and+behavioral+studies+of+acetylcholine,+amino+acid,+monoamine+and+neuropeptide+release+in+the+sheep.">Microdialysis in large unrestrained animals: neuroendocrine and behavioral studies of acetylcholine, amino acid, monoamine and neuropeptide release in the sheep.</a> Microdialysis in the Neuroscience. 7, 327-348 (1991).</li><li>Oakley, B., Schafer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental Neurobiology: A laboratory Manual. , (1978).</li><li>Okumura, T., Yamashita, Y., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+Finch+I.+A+Biological+study.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese Finch I. A Biological study.</a> Soc. Neurosci Abst. 33, (2007).</li><li>Boulton, A., Baker, G., Robinson, T., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relation+of+brain+electrical+activity+to+behavior.">The relation of brain electrical activity to behavior.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Yamashita, Y., Okumura, T., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+finch.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese finch.</a> , 101-104 (2007).</li></ol>

실험 동물의 두뇌의 실험 과학 연구소, RIKEN (RIKEN - BSI) 및 연구소의 동물 윤리위원회를 승인에 대한 지침에 따라 실시되었다.

종래의 자기 경화 아크릴 수지의 응용 프로그램에 비해 도입 기술은 몇 가지 중요한 장점이 있습니다 : 1. 직접 결합 : 셀프 경화 아크릴 잠시 동안 뼛속까지 장착할 수 있지만 그것은 뼛속까지 준수하지 않습니다. 따라서, 앵커리지 나사는 (그림 2A 참조) 마운트베이스를 수정해야합니다. 반면, 광 경화 수지는 뼈가 직접 있도록 나사가 필요하지 않습니다 (그림 2B)로 결합 수 있습니다. 따라서, 장?...

사진의 1A는 microdialysis 프로브 (A), 캡 너트 (B) 및 가이드 정맥 (C)를 보여줍니다. 이 사진에서 안내 정맥 직접 두개골을 준수 치과 수지로 둘러싸여 있습니다. 사진 B는 마약 재관류 동안 조류를 보여줍니다. 머리의 상단에, 벌금 tefron 튜브에 연결된 microdialysis 탐사선이 있습니다. B에 대한 A와 2cm에 대한 스케일 바 = 1cm, 각각. <img src="http://www.jove.com/files/249_2008_8_26_3/249_Figure-01.png" /> 그림 1 - microdialysis 프로브 (A) 및 약물 재관류 동안 조류의 사진 (B)

application of light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic experiments

만성 녹음 실험에서, 자기 치료 치과 아크릴 수지는 전극 또는 microdialysis - 프로브의 장착 기지로 사용되었습니다. 이러한 acrylics이 뼈에 결합하지 않기 때문에, 나사는 앵커로 사용되었습니다. 그러나, 핀치즈 드나 마우스 같은 작은 실험 동물에 그들의 craniums은 매우 연약하고 성공적으로 앵커를 물을 수 없습니다. 이 보고서에서는, 우리는 전극이나 만성적인 실험에 microdialysis 프로브의 설치 기반에 대한 가벼운 치료 치과 수지의 새로운 응용 프로그램을 제안합니다. 이 자료는 두개골에 직접 접착 수 있습니다. 따라서, 앵커 나사가 필요 없으며 수술 분야는 상당히 줄일 수 있습니다. 과거의 경험이 결합 효과 이상 이개월 유지 보여줍니다. 자료 고분 고 분한하고 가능한 아르 시간이 종래의 수지의 창이 몇 분 거리에 있습니다. 그러나 이러한 치과 접착제 근무 시간의 창 상당히 넓은하고 조정합니다.

Watch this Scientific Journal Video about Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments at JoVE.com

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

이 보고서에서는, 우리는 전극이나 만성적인 실험에 microdialysis 프로브의 설치 기반에 대한 가벼운 치료 치과 수지의 새로운 응용 프로그램을 제안합니다. 이 자료는 두개골에 직접 접착 수 있습니다.

만성 실험에 장착 전극 또는 Microdialysis 프로브를위한 라이트 - 치료 치과 접착제 수지의 응용

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

application-light-cured-dental-adhesive-resin-for-mounting-electrodes

Research

JoVE Journal

Biology

15.3K 조회수.  RIKEN. 이 보고서에서는, 우리는 전극이나 만성적인 실험에 microdialysis 프로브의 설치 기반에 대한 가벼운 치료 치과 수지의 새로운 응용 프로그램을 제안합니다. 이 자료는 두개골에 직접 접착 수 있습니다.

비디오: Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

전기 생리학 실험을위한 인공 Bilayers의 준비

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

연구

생물학

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

직접 레이블이 프로브를 사용하여 강력한 3D DNA 물고기

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

다층 제브라 피쉬의 장기 라이트 시트 현미경에 장착

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

총회와 &#39;전단 반지&#39;의 응용 프로그램 : 궤도, 단방향 및 정기 유체 흐름 및 전단 응력에 대한 소설 내피 모델

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

유전자 인코딩 된 형광 산화 질소의 응용 프로그램 (• NO) 단일 세포에서 NO • 신호의 실시간 이미징을위한 프로브의 geNOps,

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

마우스에 있는 정점 치주염 유도

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Systematic Assessment of Mammalian Skull Specimens for Dental and Temporomandibular Joint Pathology

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental pathology, and anatomic variations in many mammalian species. Studying the teeth and jaws of an array of species can present a challenge requiring attention to detail and understanding of a species&#39; normal anatomy. In the present article, a systematic and precise protocol for examining skull specimens is discussed that has been applied to a variety of mammals to define characteristic diseases in the oromaxillofacial region. The procedure outlined is simultaneously precise, repeatable, and adaptable to the highly differing skull and tooth shapes and anatomy across species. Specifically, specimens are examined for missing teeth, periodontal disease, endodontal disease, TMJ pathology, and anatomical variations. Results gleaned from research on museum specimens may reflect the natural history, health, and disease status of individuals and species. Furthermore, these data can inform ecological and conservation research efforts, as well as the care of captive individuals.

The present protocol describes the techniques for the systematic assessment of skull specimens to characterize anatomical and developmental variations and abnormalities of the teeth, periodontal disease, endodontal disease, and temporomandibular joint pathology.

치과 및 측두 하악 관절 병리에 대한 포유류 두개골 표본의 체계적인 평가

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental ...

치과용 치수줄기세포의 1차 배양 확립

Primary Culture of Dental Pulp Stem Cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

저자 스포트라이트: Advancing Tissue Regeneration and Disease Modeling with Dental Pulp Stem Cells(치과용 펄프 줄기 세포를 사용한 조직 재생 및 질병 모델링 발전)

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

치과용 치수줄기세포의 1차 배양

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...