このプロトコルの開発は再生医療のためのカリフォルニア工科大学（CIRM、TR1-01246）とパーキンソンアライアンスによって資金を供給された。

標準手順3（ 例えば 4ミリメートルラウンドVisipunch器で得られた）を使用して得皮膚パンチ生検は、氷の上で完全DMEM 20％FBS培地で保たれるべきである。いったんサンプルは実験室に到着した、できるだけ早く生検を処理する。 1。皮膚パンチ生検の準備 STEPS 1.1から1.3には、バイオセーフティキャビネット内で実行されるべきである <ol><li>予め調製：6ウェルプレートの各ウェルに0.1％ゼラチンを1 mlを加える。 30-60分間脇にプレートを設定します。ゼラチン溶液を吸引除去し、各ウェルに完全DMEM/20％FBS培地800μlのを追加します。ウェルの表面全体がメディアで覆われていることを確認してください。 </li><li>滅菌10センチ組織培養皿の蓋を反転し、蓋の中央にDMEM / 20％FBS培地1.5mlのを追加し、血清ピペットの先端とメディアドロップを広げる。 </li><li>滅菌ピンセットを使用して、場所T彼は皮膚皿にメディアで作品を生検。 </li></ol> 2。皮膚パンチ生検の解剖 STEPS 9 2.1から2.2、水平層流フード内で実行されるべきである <ol><li>反転した蓋の上に10センチ皿の底の部分を置き、層流フード内解剖顕微鏡に皮膚生検を転送します。 </li><li>場所で生検を保持するために、1メスを使用し、一つの方向に転動でカットすることが第二のメス等分にピースをカットして、シャープなエッジを持つ12-15均等なサイズの部分に4ミリラウンド皮膚生検を解剖。不規則なエッジを持つ個貧しいアタッチメント/細胞伸長に貢献しています。 </li></ol> 3。組織培養プレートに解剖皮膚生検個の譲渡 STEPSは、3.1から3.6バイオセーフティキャビネット内で実行されるべきである <ol><li> 10センチ組織培養皿の底部を配置する反転蓋の上に、とバイオセーフティキャビネットにに戻っ皿を移す。 </li><li>ドライウェルに800μlのではなく、を含む調製された6ウェルプレートの各ウェルに尖った鉗子、場所2-3生検部分を使用して。井戸の底に付着する部分を取得するにはタップまたはスライドモーションを使用してください。メスは、鉗子からの生の部分を除去するのに有用である。 </li><li> 37℃のインキュベーター内で6ウェルプレートを置きます。メディアコーティングのフィルムは最初の週のために井戸の底があることを確認するために、毎日監視し、任意の蒸発メディアを交換するには2日ごとに〜200μLを加える。 </li><li> 1週間後、2〜3日ごとに完全なDMEM/20％FBS 2mlのにメディアの量を増加させ、メディアを変更してください。 </li><li>一度線維芽細胞は線維芽細胞が2X T75フラスコ（継代1）に入っても、トリプシン処理及び通路6ウェルプレートの縁に達している点に、各ウェルに合流されています。組織片も同様に転送することができる。彼らは、Oを添付せず、洗浄することが次のメディア変更時UT。 </li><li>一度線維芽細胞がコンフルエントあり、バイアルあたり1×10 6細胞/ mlで完全DMEM培地プラス10％のDMSOで凍結、3X T175フラスコ（通路2）に転送します。 </li></ol> 4。免疫染色を通してキャラ線維芽細胞を<ol><li> 8ウェルチャンバースライドでの培養線維芽細胞は、ゼラチンでコーティング。 </li><li>細胞が80％コンフルエントにあるとき、各ウェルから培地を吸引。 </li><li>室温で10分間4％パラホルムアルデヒドで細胞を固定する。 </li><li> 1X PBSでウェルを3回洗浄します。 </li><li> 0.3％150μlのトリトンX-100、室温で5分間（PBS中）で細胞を透過性。 </li><li> 1X PBSでウェルを3回洗浄します。 </li><li>室温で1時間、200μlのブロッキング溶液（PBS + 5％正常ヤギ血清（ベクター、S-1000））を加える。 </li><li>ブロッキング溶液でアンチSERPHIN-1（抗ウサギ、MAB、シグマ、S5950-200μL）の1:250希釈を準備します。 </li><li> Aブロッキング溶液をspirate、とAntiSERPINH-1の1:250希釈150μlのを追加します。室温で1〜2時間でインキュベートする、または4℃で一晩。 </li><li> 1X PBSでウェルを3回洗浄します。 </li><li>ブロッキング溶液で1:200ヤギ抗ウサギモノクローナル抗体（Alexaの弗素ヤギ抗ウサギIgG（H + L）、Invitrogen社、A11008）を準備する。 </li><li>各ウェルに1:200ヤギ抗ウサギモノクローナル抗体150μlのを追加します。室温で1時間、暗所で、インキュベートする。 </li><li> 1xPBSで一回井戸を洗浄します。 </li><li> PBSを吸引し、慎重にだけスライドを明らかにするためにチャンバーを持ち上げ。 </li><li> DAPI（ベクター、H-1200）を含む培地を取り付ける2-3滴でスライドをマウントします。 </li><li>蛍光顕微鏡下でスライドを分析します。 </li></ol>

<ol>
	<li>Nguyen, H. N., 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=LRRK2+mutant+iPSC-derived+DA+neurons+demonstrate+increased+susceptibility+to+oxidative+stress.">LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress.</a> Cell Stem Cell. 8, 267-280 (2011).</li><li>Mak, S. K., Tewari, D., Tetrud, J. W., Langston, J. W., Schule, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+skin+fibroblasts+from+a+Parkinson's+disease+patient+with+an+alpha-synuclein+triplication.">Mitochondrial dysfunction in skin fibroblasts from a Parkinson's disease patient with an alpha-synuclein triplication.</a> Journal of Parkinson's Disease. 1, 175-183 (2011).</li><li>Punch Zuber, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=biopsy+of+the+skin.">biopsy of the skin.</a> Am. Fam. Physician. 65, 1155-1164 (2002).</li><li>Takashima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+fibroblast+cultures.">Establishment of fibroblast cultures.</a> Curr. Protoc. Cell Biol. Chapter 2, Unit 2, 1 (2001).</li><li>Kuroda, K., Tajima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP47+is+a+useful+marker+for+skin+fibroblasts+in+formalin-fixed,+paraffin+embedded+tissue+specimens.">HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin embedded tissue specimens.</a> J. Cutan. Pathol. , 241-246 (2004).</li></ol>

著者らは、開示することは何もありません。

このプロトコルを使用して、皮膚線維芽細胞の比較的純粋な培養物を得ることができる。線維芽細胞は楕円形の細胞核へのラウンド細長い紡錘状細胞体の特徴的な形態学的特徴を持っており、線維芽細胞が整列し、バンドル時合流で育つ。他の細胞集団などのケラチノサイトが追加料金と成長因子を必要とするか、有糸分裂活性が低いのに対し、メディアは線維芽細胞の成長をサポー?...

<table border="1">
<tbody>
 <tr>
 <td>Name of Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Dissecting microscope</td>
 <td>Leica</td>
 <td>S6E</td>
 </tr>
 <tr>
 <td>10 cm Tissue Culture Petri dish</td>
 <td>VWR</td>
 <td>25382-166</td>
 </tr>
 <tr>
 <td>6-well Tissue Culture plate </td>
 <td>VWR</td>
 <td>73520-906</td>
 </tr>
 <tr>
 <td>Stainless steel disposable sterile scalpels (2), blade No#15, Miltex</td>
 <td>VWR</td>
 <td>21909-660</td>
 </tr>
 <tr>
 <td>Sterile pointed-tip forceps</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 mL Conical tubes</td>
 <td>VWR</td>
 <td>21008-940</td>
 </tr>
 <tr>
 <td>2 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-894</td>
 </tr>
 <tr>
 <td>5 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-896</td>
 </tr>
 <tr>
 <td>10 mL Serological Pipettes</td>
 <td>VWR</td>
 <td>89130-898</td>
 </tr>
 <tr>
 <td>Pasteur Pipettes</td>
 <td>VWR</td>
 <td>14672-380</td>
 </tr>
 </tbody>
</table>
Table A. Table of specific reagents and equipments.
 <table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent</td>
 <td>Company </td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>1X DMEM: High Glucose</td>
 <td>Invitrogen/Gibco</td>
 <td>11960-069</td>
 </tr>
 <tr>
 <td>20% Fetal Bovine Serum</td>
 <td>Invitrogen/Gibco</td>
 <td>26140-079</td>
 </tr>
 <tr>
 <td>1X L-Glutamine</td>
 <td>Invitrogen/Gibco</td>
 <td>25030-164</td>
 </tr>
 <tr>
 <td>1X MEM Non-essential Amino Acids </td>
 <td>Invitrogen/Gibco</td>
 <td>11140-076</td>
 </tr>
 <tr>
 <td>1X Penicillin/Streptomycin</td>
 <td>Invitrogen/Gibco</td>
 <td>15140-163</td>
 </tr>
 </tbody>
 </table>
Table B. Reagents.

skin punch biopsy explant culture for derivation of primary human fibroblasts

疾患を有する個体由来の組織および細胞株は、疾患関連細胞表現型を研究するための理想的な供給源である。このプロトコルにおける患者由来の線維芽細胞を正常にモデル疾患1に人工多能性幹細胞の誘導に用いられてきた。これらの線維芽細胞の初期の通路はまた、特定の疾患経路、機構2およびそれに続く薬剤スクリーニング手法を研究するために、細胞ベースの機能アッセイに使用することができる。酵素手続き上提示プロトコルの利点は、1）酵素処理を用いた高齢患者、パーキンソン病、2に罹患例えば患者）より挑戦的な方法論の上に技術的に簡単なアプローチから派生した組織の少量からの技術の再現性、および3ですの）時間を考慮：このプロトコルは、15〜20分かかりますし、生検到着した直後に行うことができます。酵素処理は、4時間までかかるとの問題を抱えていることができます過剰消化、細胞の生存と正しく処理されていない細胞のその後のアタッチメントの削減。このプロトコルは、4mmのヒト皮膚線維芽細胞培養物の誘導のための生検の切開及び調製を記載し、患者由来の組織サンプルを扱うときに重要である、非常に高い成功率を有している。この文化では、ケラチノサイトは、調製後最初の一週間以内に生検組​​織の外に移行します。線維芽細胞は7-10日ケラチノサイトの最初の伸長後に表示されます。 20％FBSを添加したDMEM高グルコース培地はケラチノサイトおよび線維芽細胞上の線維芽細胞の成長がケラチノサイトの過剰増殖します好む。 2継代後ケラチノサイトは線維芽細胞マーカーSERPINH1（HSP-47）を発現する、比較的均質な線維芽細胞培養の結果から希釈されています。このアプローチを使用して、15から20000000線維芽細胞は、細胞バンキングのため4-8週間のうちに誘導することができる。皮膚切開は15-20分ほどかかり、その後、細胞を一日一回監視され顕微鏡下で、メディアは2〜3日ごとに細胞の付着と伸長した後に変更されます。

ケラチノサイトは解剖後の48時間とすぐに生検片の外に成長しています。最初の線維芽細胞の伸長は、処理後1週間程度を観察することができる。井戸がコンフルエントに達した後3 150センチメートルフラスコ（T150）と細胞に到達するには、2つ以上の通路は凍結保存されているため、線維芽細胞を継代する。我々はこの方法で15から20000000までのセルを生成します。線維芽細胞は、小胞体に局?...

Watch this Scientific Journal Video about Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts at JoVE.com

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

ヒト皮膚パンチ生検からの線維芽細胞移植片培養プロトコルは、低継代数で約15〜20万人の細胞バンキングのため4-8週間以内に皮膚細胞を導出するために、技術的に堅牢かつ簡単な方法です。

プライマリーヒト線維芽細胞の導出のために皮膚パンチ生検植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

skin-punch-biopsy-explant-culture-for-derivation-primary-human

Research

JoVE Journal

Medicine

40.8K ビュー.  The Parkinson's Institute. ヒト皮膚パンチ生検からの線維芽細胞移植片培養プロトコルは、低継代数で約15〜20万人の細胞バンキングのため4-8週間以内に皮膚細胞を導出するために、技術的に堅牢かつ簡単な方法です。

ビデオ: Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

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.

ヒト乳腺線維芽細胞からの癌関連線維芽細胞の実験的生成（CAFS）

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

医学

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

パーキンソン病の分野におけるミトコンドリアの表現型を監視するための主要ヒト線維芽細胞の使用

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

プライマリマウス大腸腫瘍のin vitroオルガノイド培養で

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

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.

初代ヒト骨髄およびその悪性腫瘍を研究する三次元組織培養モデル

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

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

人間の前庭神経鞘腫の初代培養

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&amp;#246;m Technique

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

変更されたベルイストローム法を用いたヒト骨格筋生検手順

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

全組織生検からヒト上皮EnteroidsとColonoidsの確立

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人間<em&gt;外側広筋</emワイル・ブレークスリーConchotomeを使用&gt;骨格筋生検

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

スキンマイクログラフト世代の新しい脱凝集法後の組織性状

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

光シート顕微鏡による劇症心筋炎のマウスモデルにおける白血球浸潤の定量的可視化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...