The authors acknowledge financial support from the Victorian Government&#8217;s Operational Infrastructure Support Program.

注：胎盤用語選択科目帝王切開の設定に合わせて、シングルトン健康妊娠から収集する必要があります。書かれ、インフォームドコンセントは、胎盤の収集のために与えられるべきである。あなたの関連する人間の研究倫理委員会の承認のヒト組織のすべての収集と利用をする必要があります。 羊膜上皮細胞の単離<ol style="margin-left: 40px;"><li>クラスII生物学的安全キャビネット内で無菌の表面に胎盤を置きます。 </li><li>無菌ハンクスの平衡塩類溶液（HBSS）を用いて、胎盤の表面及び膜からできるだけ多くの血液を洗浄する。 </li><li>臍帯を上にして置かれ、胎盤では、機械的に羊膜と絨毛膜の膜の外縁を分離する。 </li><li>分離後は、手動で臍帯に向けて、膜のエッジの周りの作業、胎盤の絨毛膜膜から羊膜を取り除く。 注：carefこと羊膜から絨毛膜を汚染する任意の部分を削除するには、UL。 </li><li>無菌のハサミを使用して、250ミリリットルHBSSを含有する容器を密封された500ミリリットル中に臍帯と場所のベースから約2cmの羊膜をカット。 </li><li>羊膜膜を含む密閉ボトルを振ることにより徹底的に洗ってください。 </li><li>滅菌ピンセットを使用して収集容器から羊膜膜を外し、15センチメートルペトリ皿に置く。 </li><li>流血や破れの部分を破棄し、（ピンセットで垂直に保持時）約5cm、長片に羊膜をカット。 </li><li> 500ミリリットル内の場所は、コンテナを密封し、250ミリリットルHBSS約3〜5倍または羊膜膜が無汚染血液と半透明になるまで洗浄した。 注：任意に存在する血液の酵素消化溶液の効率を低下させることができる。 </li><li>に注意しながら、溶液を消化する酵素を50 mlを入れた容器に羊膜のすべての部分を転送するHBSSのキャリーオーバーを最小限に抑える。 </li><li>残りの血液を除去するために穏やかに攪拌しながら15分間37℃の水浴または暖かい部屋でソリューションを消化酵素の中で羊膜断片をインキュベートする。 </li><li>新しい滅菌容器内の溶液と場所を消化酵素のから羊膜片を除去します。 </li><li>羊膜膜へのソリューションを消化し、水浴または穏やかに振盪（最初の消化）と暖かい部屋で60分間37℃でインキュベート新鮮な酵素の100ミリリットルを追加します。 </li><li>羊膜膜片を取り出し、酵素による、膜の各部分から排水するソリューションを消化できるように、新しいコンテナ世話に入れる。 注：これは、容器の内側縁に沿って羊膜片をドラッグすることによって達成することができる。 </li><li>羊膜膜片に対する解決策を消化し、穏やかに振盪（第二の消化）を用いて37℃の水浴または暖かい部屋で60分間インキュベートする新鮮な酵素の100ミリリットルを追加します。 </li><li>セルを渡しsuspensiステップ1.14から10分間千×gで70μmのフィルターと遠心分離機を経て上に。 </li><li>削除し、上清を捨て、および1mg / mlの大豆ベースの酵素阻害剤を含む4ミリリットルHBSSで細胞ペレットを再懸濁します。穏やかに単一細胞懸濁液を得た1mlピペットで繰り返しピペット。 注：第消化物からの細胞は、同様の処理段階になるまで、この段階で、細胞を氷上に放置することができる。 </li><li>羊膜膜片を取り出し、酵素による、膜の各部分から排水するソリューションを消化できるように、新しいコンテナ世話に入れる。 注：これは、容器の内側縁に沿って羊膜片をドラッグすることによって達成することができる。 </li><li> 10分間の千×gで70μmのフィルターと遠心分離機を通してステップ1.18から細胞懸濁液を渡します。 </li><li>削除し、上清を捨て、および1mg / mlの大豆ベースの酵素阻害剤を含む4ミリリットルHBSSで細胞ペレットを再懸濁します。静かに1ミリリットルpで繰り返しピペット単一細胞懸濁液を得るipette。 </li><li> 2つのダイジェストまたは代わりのプール細胞は、細胞数の後まで分離した細胞ダイジェストを保つ。 注：これは高い生存細胞と、潜在的に低い生存性細胞の混合を防ぐことができます。 </li><li> 10〜20ミリリットルの最終容量にHBSSを追加し、生存能力を測定するためにトリパンブルーを用いて細胞数を行う。 </li><li>フローサイトメトリーによる上皮細胞表現型を確認してください。抗EpCAM-PEを用いて適切な抗体カクテルを作る（1：2希釈）抗CD90-PeCy5（1：250）および抗CD105-APC（1：100）HBSS中。 </li><li> 25×10 6細胞/ mlの濃度で一つまたは抗体カクテルの両方の消化物からの細胞のサブセットを再懸濁する。 </li><li>フローサイトメトリー、ステインアイソタイプコントロールを含む抗体カクテルと1×10 6細胞（例えば。のIgG 1アイソタイプコントロール-PE +抗CD90-PeCy5 +抗CD105-APC）。セットアップフローサイトメーター用の染色されていない〜1×10 6細胞を保管してください。 </li><li>抗体で細胞をインキュベート60分間4℃でカクテルをお楽しみいただけます。 </li><li>洗浄細胞は、フローサイトメトリーのために、1ml当たり5から10000000個の細胞でHBSS再懸濁で3倍。 </li><li>フローサイトメトリーとノート結果を実行します。細胞分離株（のEpCAM陽性およびCD90 / CD105陰性細胞の割合）の純度は、それぞれ90〜95％と&lt;1％であることが期待される。 </li><li>最終的なアプリケーションによって決定されるよう、適切な品質管理やリリース基準のテストのために取って細胞の適切な数を設定します。 注：これは、疾患/ウイルスのスクリーニング、または他の安全性又は生物学的基準を含むことができる。 </li><li>残りの細胞では、低温保存プロトコールに従うか、あるいは（培養工程3.6への直接）文化に直接配置します。 </li></ol> HAECを2.凍結保存<ol style="margin-left: 40px;"><li>トリパンブルー排除を使用してデータまたは手動カウント、フローサイトメトリーを用いて細胞数および生存率を決定します。 </li><li> 5分間700×gで遠心分離する。 </li><li>再懸濁細胞動物性製品の無凍結保存培地1ml当たり1から10000000細胞間の秒。 </li><li> O-環状凍結保存バイアルに細胞の適切な量をピペット冷却℃/分まで-80°C〜が達成され、およそ1を発生する装置を凍結し、制御された速度に入れる。 </li><li>長期保存のために液体窒素中にバイアルを移す。 </li></ol>凍結保存HAECを3.解凍と文化<ol style="margin-left: 40px;"><li>液体窒素から凍結保存バイアルを取り出し、氷上に置きます。 注：転送の場合のみ - 次のステップに急速に進んでください。 </li><li>氷が残る唯一の小片（〜5mm角）になるまで37℃で急速に凍結バイアルを解凍する。 注：細胞溶液は、凍結保存培地中のDMSOの毒性効果を減少させるために加温しないことが重要である。 </li><li>冷（1~4℃）で無血清培地に凍結保存培地を10倍に希釈し、適当な大きさに滴下D管やコンテナ。 </li><li> RTで5分間700×gで遠心分離する。 </li><li> 1mlあたり100万個の細胞のおよその濃度を、無血清培地中で再懸濁し、。トリパンブルー排除で自動化または手動の計数を用いて細胞数および生存率を決定します。予想される生存解凍後は、事前に凍結保存の生存率よりも5〜10％減となります。 </li><li>代わりにプレート標準的な細胞培養処理プラスチックウェア上の細胞、または、私はコート培養表面に型コラーゲンを使用しています。 </li><li> I型コラーゲンコーティングについては、以下のプロトコルに従う<ol><li>ヒト胎盤（25ミリリットル脱イオン水と50μlの氷酢酸と15 mgのコラーゲン）から酸可溶性I型コラーゲンを準備します。 </li><li>パラフィルムでビーカーを覆い、コラーゲン鎖が溶解するまで37℃で適度に撹拌した。 </li><li> 0.22μmのフィルターを用いて無菌濾過コラーゲン溶液。 </li><li>脱イオン水でろ過したコラーゲン株式1:10に希釈する。この希釈された株式は、作業conceですプラスチックと膜表面をコーティングするためのntrationソリューション。 </li><li> 2〜8℃、37℃、またはO / NでのコラーゲンRTで2時間コートプラスチック、ガラス、膜の表面を。 </li><li>コー​​ティングされた表面から余分な流体を除去し、それがO / Nを乾燥させます。 </li><li>細胞および培地を導入する前に無菌の組織培養グレードの水またはHBSSで洗い流してください。 </li></ol></li><li>プレート無血清培地中で25000細胞/ cm 2の密度でHAECを、そして、添付ファイルが72〜96時間起こさせる。 </li><li>添付ファイルの後、慎重に吸引し、無血清培地の適切な音量2〜3日ごとに交換することによってメディアを変更。 </li><li>以下のプロトコルを使用して継代細胞<ol><li>吸引し、細胞培養培地および滅菌HBSSの1倍容量で洗浄</li><li>細胞は、細胞培養表面から離脱観察されるまで、酵素の1倍体積を加える溶液を消化し、5〜15分間インキュベートし、または。 </li><li> CEのを確実にする、細胞を含む酵素消化液の収集LLSを繰り返し、細胞培養表面に対する酵素消化物溶液をピペッティングすることにより細胞培養表面から剥がれた</li><li> 5分間700×gで遠心分離する。 </li><li> 1mg / mlの大豆ベースの酵素阻害剤を含有するHBSSの適切な体積で細胞ペレットを再懸濁する。穏やかに単一細胞懸濁液を得るために、1ミリリットルピペットで繰り返しピペット。 </li><li> 100万個の細胞/ mlのおおよその濃度になるように無血清培地を加える。トリパンブルー排除で自動化または手動の計数を用いて細胞数および生存率を決定します。 </li><li>プレート無血清培地中で25000細胞/ cm 2の密度でHAECを、そして、標準的な培養方法を続行。 </li></ol></li></ol>

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+mesenchymal+stem+cells+obtained+from+different+human+tissues.">Comparison of mesenchymal stem cells obtained from different human tissues.</a> Bull Exp Biol Med. 139 (4), 504-509 (2005).</li><li>Park, A., 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=.">.</a> Newborn Stem Cells: Identity, Function, and Clinical Potential. , 119-137 (2013).</li></ol>

The authors have nothing to disclose.

この方法の成功に重要な影響を与えることができるいくつかの重要なパラメータがある。 HAECを単離する前に、最大3時間胎盤または羊膜の貯蔵はロジスティックまたはスケジューリングの目的のために望ましいことがある、しかしそれは、組織ができるだけ早く処理されることをお勧めします。組織を保存する場合は、ストレージが羊膜膜の解剖と洗浄後に実施することをお勧めします。羊?...

そのような胎盤などの周産期の供給源に由来する細胞は、胎盤膜、臍帯および羊水は再生医療1,2のための細胞の潜在的な供給源として研究者や臨床医から注目を集めている。この関心の理由は、これらの細胞型すべてが可塑性および免疫調節機能3、彼らの潜在的な治療への応用の基礎となる性質をある程度持っていることである。 HAECを回生細胞物質の豊富な潜在的な源を提供する、用語または早産羊膜4に由来することができる不均一な上皮細胞集団である。細胞療法として魅力HAECをを作る特性は、それらの多能、低い免疫原性、および抗炎症性を含む。 HAECをはcardiom（中胚葉系統に分化する能力、in vitroおよびin vivoの両方で非常に多能性であることが判明しているyocytes、筋細胞、骨細胞、脂肪細胞）、内胚葉系統（膵臓細胞、肝細胞、肺細胞）および外胚葉系統（毛髪、皮膚、神経細胞とアストロサイト）5-10。 安心させる、彼らの多分化のHAECをもかかわらず、腫瘍を形成またはin vivoで腫瘍の発達を促進するためのいずれか表示されません。さらに、HAECを、クラスIIヒト白血球抗原（HLAは）8の低いレベルで発現する、特権免疫がある。このプロパティは、おそらく免疫適格サル、ウサギ、モルモット、ラット、およびブタ11-13を用いた研究において実証されるように、同種および異種移植後の免疫拒絶を回避する能力の基礎となる。 HAECを、強力な免疫調節および免疫抑制のプロパティを表示し、したがって、自己免疫疾患の治療における潜在的な臨床応用のための重要な実用的な利点を提供する。 HAECをは、先天性および適応免疫システムの両方に免疫調節機能を発揮すると考えられている。上のメカニズムのeは、免疫調節因子の14の分泌を介して行われ、提案した。 前臨床動物疾患モデルでHAECを、現在のアプリケーションは、脳卒中、多発性硬化症、肝疾患、糖尿病、慢性及び急性の肺疾患の治療が含まれる。研究者は、彼らの独特の特性に起因する脳卒中後の脳の炎症を処置するためにHAECをを使用することに関心を示している。 HAECを、彼らは、生着までの60日間生存、ニューロンへの分化、炎症を減少させ、神経疾患15の動物モデルにおいて、損傷した中枢神経系組織の再生を促進することができ、血液脳関門を通過することができるという証拠がある。 HAECを、多発性硬化症の発症および進行に寄与する複数の病理学的経路を標的とし、逆転する機能を提供します。例えば、前臨床動物試験からの結果は、HAECを強く免疫抑制性であることを示唆していると潜在的に末梢免疫寛容を誘導し、継続的な炎症反応を逆にすることができます。 HAECをまた、インビボで神経細胞に分化し、神経栄養因子16の広大な配列の分泌を介して内因性の神経再生を増強する能力を有することが示されている。 ヒト及びげっ歯類羊膜上皮細胞は、既に、動物モデルにおける肝疾患の治療のためのそれらの治療有効性を実証している。縮小肝細胞アポトーシスに伴う肝臓中の生存HAECを生着に肝疾患、HAEC移植リードの四塩化炭素による損傷誘発モデルにおいて、肝線維症、炎症および17を減少させた。 HAECを、インスリンとグルコーストランスポーターなどの表現膵臓の要因に刺激することができる。いくつかの研究は、糖尿病マウス18で血中グルコースレベルを復元するHAECをする可能性を検討した。投与したマウスでは、HAECをは、両方の動物の体重および血中グルコースレベルは、細胞の注射後に正常レベルに低下した。これらの研究は、糖尿病の治療のためのHAECを使用するための強力なケースを提示する。 HAECを、成人および新生児モデル19の両方で実験的な急性および慢性の肺損傷の防止と修復において実績のある役割を担っている。これらの研究は、HAECをが嚢胞性線維症膜貫通コンダクタンスレギュレータ（CFTR）、嚢胞性線維症を有する患者20に変異しているイオンチャネルを含む複数の肺癌関連タンパク質を発現する機能的な肺上皮細胞にin vitroで分化することがわかった。 HAECを負傷が成人および新生児の肺に送達された場合も、それらは、好中球、マクロファージおよびリンパ球21-23を含む肺白血球補充を、還元、宿主免疫細胞の調節を介して、それらの修復効果を発揮する。 彼らの豊富を考えると、安全記録、および複数の疾患のための実証済みの臨床応用、HAECをを使用した臨床試験は避けられない。臨床治験にHAEC療法の翻訳を加速させることを目標に、私たちは現在の適正製造基準（cGMPの）ガイドラインに従って動物製品の無試薬を用いて、臨床試験のための適当な方法で培養HAECをし、分離し凍結保存とする方法を開発。 私たちは、このプロトコルを私たちは動物由来の試薬​​6を使用してHAECをを分離することに成功し使用していた以前に公開されたプロトコルをベース。我々は、動物製品を含まない試薬で動物由来の製品を置き換えるために、元のプロトコルを変更し、その後の最適化は、細胞収量、生存率および純度を最適化するために行った。私たちの目標は、ヒト臨床試験用電池製造のための規制基準に準拠したいプロトコルを開発することであった。

<table border="0" cellpadding="0" cellspacing="0" width="641">
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			<td>356550</td>
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			<td>352096</td>
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			<td></td>
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			<td>A451-4&#160;</td>
			<td></td>
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			<td></td>
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	</tbody>
</table>

isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

この手順が正しく実行されると、1.2億HAECを、平均収量は80から160000000セルの典型的な範囲で、期待されるべき。これらの収率で、83±4％の平均生存率が期待できる。臨床的方法で増加し、平均収率およびわずかに低い生存率に起因血清タンパク質の欠如のために、おそらく、動物由来の製品よりも高いトリプシン活性に起因する、とすることができる。単離されたHAECをは&lt;1％CD90、CD105間葉?...

Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

臨床応用のためのヒト羊膜上皮細胞の単離、凍結保存と文化

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

isolation-cryopreservation-culture-human-amnion-epithelial-cells-for

Research

JoVE Journal

Medicine

13.8K ビュー.  Wake Forest University Health Sciences. We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines. 

ビデオ: Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

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.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

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

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

ヒト胎児膵臓細胞クラスターの単離、培養、およびイメージング

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

臨床および前臨床研究用途のための半自動循環腫瘍細胞（CTC）アッセイの適応

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

腫瘍特異的内皮細胞の単離および培養拡大

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

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

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

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

ヒト下垂体腺腫細胞の三次元アルギン酸ビーズ文化

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

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

犬の動脈と静脈からの初代内皮細胞の単離および培養

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

ひと腎臓血管周囲間質細胞の新規臨床等級分離法

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

根治的前立腺切除術後の勃起不全治療のための脂肪由来再生細胞の単離

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

睡眠美容トランスポゾントランスポゾントランスフェクトヒト網膜色素上皮細胞における酸化ストレスの誘導と分析

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...