著者は、技術支援のために博士は東軒家に感謝したいと思います。この記事に表示される画像の一部は、博士マルコムS.スタインバーグ、分子生物学科、プリンストン大学と共同でいました。著者はまた、彼らの寛大な支援のために国防前立腺癌研究プログラム（助成金のPC - 030482およびPC - 991552）とNCI / NIH（助成R01CA118755）の部門に感謝の意を表します。

1。単一の細胞懸濁液の調製<ol><li>接着細胞の培養は90％のコンフルエンスまで増殖されるべきである、whereupon単層をPBSで2回リンスする必要があります。よく排水後、0.05％トリプシン- 1mMのEDTAの2 MLSを（100 mmプレートの場合）追加し、37℃での細胞がデタッチされるまで。完全培地の2 MLSを追加することにより、トリプシン処理を停止し、静かに、細胞が懸濁液になるまで混合物を砕いて粉にするために5 mlのピ​​ペットを使用してください。 15 mlコニカルチューブに細胞を移す。 </li><li>室温で5分間に10 mg / mlのDNase株式とインキュベートの40μlを添加する。 5分間200 xgで軽くボルテックスして、遠心。 </li><li>上清を捨て、1mlの完全な組織培養の培地でペレットを洗浄。繰り返しますが、その後、完全な組織培養培地の2 MLSで細胞を懸濁します。 </li><li>血球計算盤、または自動細胞計数装置を用いて細胞をカウントし、2.5 × 10 6細胞/ mlに濃度を調整する。このデモではBioRad社TC10自動細胞計数装置を用いた。 </li></ol> 2。ハンギングドロップの形成。 <ol><li> 60 mmの組織培養皿と皿の底の場所のPBS 5mlのをから蓋を外します。これは、水和室として機能します。 </li><li>蓋を逆さにして蓋の底部上に10μlの滴を堆積させるために20μlのピペッターを使用してください。触れないでするように十分離れてドロップが配置されていることを確認してください。皿ごとに少なくとも20滴を配置することが可能です。 </li><li> 37 PBS -満ちボトムチャンバーとインキュベート℃/ 5％CO 2 /湿度95％の上にふたを反転させる、毎日の低下を監視し、細胞シートまたは凝集体のいずれかが形成するまでインキュベートする。ステレオ顕微鏡は、凝集体形成を評価するために使用することができます。 </li><li>一度シートの形、それらが回転楕円体の形になるまで℃、5％CO 2を完全培地の3のMLSを含む丸底ガラスシェーカーフラスコに移し、37で振とう水浴中でインキュベートすることができます。 </li></ol> 3。ノート<ol><li>セルのサイズに応じて、濃度が上下調整する必要があります。 </li><li>細胞は、膜はハンギングドロップ形成する前に蛍光色素を吸蔵で染色することができます。 </li><li>二つの異なる細胞型を差動でぶら下がっ滴の形成前に1:1（またはその他）の割合で染色し、混在させることができます。これらは遺伝的に特定の接着剤の署名を持つように設計癌と間質細胞、胚組織、または細胞であり得る。 </li><li>細胞は、カドヘリンの機能を維持するために0.05％トリプシン/ 2 mMのカルシウムを使用して組織培養皿から剥離することができます。 </li><li>実験によっては、用紙サイズはいずれかで測定することができる、または集計は機械的な試験や生化学的または分子解析のために使用することができます。 </li></ol> 4。代表的な結果： シートまたは回転楕円体の形成は、通常24時間以内に起こるが、細胞の種類に応じて時間がかかることがあります。図1は、未治療前立腺癌MAT - LyLu（MLL）ドロップ文化を吊り下げ中の細胞（図1A）または25μmのMEK阻害剤PD98059で処理した細胞のインキュベーションの18時間後に撮影した画像を表します。細胞シートの著しい圧縮におけるそのMEKiの治療の結果に注意してください。圧縮は10-20のサイズ（またはそれ以上）滴を吊り下げ、治療群間の平均サイズを比較して測定することによって定量することができます。我々はピクセル単位で画像全体の面積を明らかにするために、粒子の分析を適用するとすぐに我々は一般的に、バイナリモードに画像を、各画像を閾値変換することにより、ImageJのソフトウェアを使用して画像を分析する。これは、その後、簡単に平方ミクロンに変換することができます。図2は、未処理とMEKi処理MLL細胞のサイズの比較を表しています。スチューデントt -検定（P &lt;0.0001）で比較して、前述したように、MEKiの治療は大幅にシートサイズを減らします。この比較のために、未処理及び処理した細胞の各10凝集体を測定した。図3は、ドロップの文化をぶら下げで24時間とシェーカーバスで2日後に形成されたニワトリ胚肝臓の回転楕円体を表しています。 ハンギングドロップアッセイはまた、採択された空間的位置を明らかにするために複数の細胞型を含むように変更することができます。例えば、ニワトリ胚の肝細胞は、蛍光膜 - インターカレート色素で染色し、別の蛍光マーカーで染色されているニワトリ胚心臓の細胞と混合した、単離することができる。混在したままになることではなく、図4Aに示すように、肝臓や心臓の細胞は一から別の&quot;アウト並べ替え&quot;と心臓の細胞が肝細胞に包まれている球の内、球の構成を採用する傾向がある。この設定は、肝臓と心臓の細胞間の細胞間接着の違いによって説明できる。この概念は、膵島細胞の組織5用とi同一の2つの細胞集団の間で細胞選別のため、両生類の3と硬骨魚類4胚の胚性生殖層の組織との間の細胞ソートの動作を説明するために使用されている差動接着仮説に成文化されNカドヘリン（6および図4B）の同じ種類の発現レベル以外のすべての点。この結果は、細胞集団の間の接着の単純な違いをエンジニアリング方法を実演するために特に重要なのは、例えば、臓器の構造の発生につながる可能性、膵島5。 <img src="/files/ftp_upload/2720/2720fig1.jpg" alt="図1" /> 図1のどちらかが存在しない（A）または存在下で18時間点滴培養をぶら下げでインキュベートした細胞の画像（B）のMEK阻害剤PD98059の25μmの。画像は、ニコンのEclipse TE 300落射蛍光顕微鏡とPhotometrics CoolSnap ESデジタルカメラを使用して捕獲された。 <img src="/files/ftp_upload/2720/2720fig2.jpg" alt="図2" /> 図2。ラット前立腺癌MLL細胞のMEKi誘発性圧縮の定量。上記のような凝集体サイズを決定したとすぐに未処理とMEKi処理MLL細胞のハンギングドロップ培養を18時間インキュベートした。凝集体サイズの統計的分析はスチューデントt検定によって行った。アスタリスクは、未処理とMEKi処理細胞間の集約サイズで統計学的有意差（P &lt;0.0001）を表し、その有意に小さく、よりコンパクトな集合体でMEKi処理の結果を示唆している。 <img src="/files/ftp_upload/2720/2720fig3.jpg" alt="図3" /> 図3。インキュベーターにして18時間点滴培養を吊り下げ/ 48時間風呂を振りにニワトリ胚肝細胞をインキュベートすることによって形成された回転楕円体。この画像は、Nikon SMZ800ステレオ顕微鏡とソニーブラックCCDカメラを用いて捕獲された。 <img src="/files/ftp_upload/2720/2720fig4.jpg" alt="図4" /> 図4。ドロップの文化を吊るすには、異なる種類の細胞の間にソートアウト挙動を明らかにする。パネルでは、ニワトリ胚の肝細胞がPKH - 2膜インターカレート色素で染色し、PKH - 26ステンドニワトリ胚心臓の細胞と同じ割合で混合。パネルBでは、二つのN -カドヘリン-トランスフェクトL細胞クローン（LN4とLN2a）、2.4:1の比率で、その表面でのN - CADを表現するには、また、蛍光膜インターカレート色素PKH - 2、PKH - 26で染色した、（Sigma - Aldrich）を、等しい割合で混合し、滴7を吊り下げて培養した。文化の中で18時間後、細胞シートは、振盪フラスコに移し、さらに48時間インキュベートした。集計がに2％パラホルムアルデヒドで固定したリン酸緩衝生理食塩水とニコンオプチフォト- 2顕微鏡に接続されているバイオラッドMRC600走査型レーザー共焦点顕微鏡システムで表示。緑と赤の両方のチャンネルからの光学切片を採取したとVital Imagesはボクセルビューウルトラのイメージングソフトウェアを搭載したシリコングラフィックスクリムゾンVGXのワークステーションは、両方のチャンネルに偽色を割り当てることや画像をマージするために、生成された解剖学的構成を明らかに使用されていました。（）擬似カラー青の肝細胞（8から）で心臓の細胞（ここで疑似カラーの黄色の）の包囲を示す集約を通じて、共焦点光学セクション。 N -カドヘリン（赤）（6）の低レベルを発現することにより、高N -カドヘリンを発現する細胞（緑色）の集合体を実証包囲から（B）共焦点光学部。

<ol>
	<li>Cukierman, E., Pankov, R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+interactions+with+three-dimensional+matrices.">Cell interactions with three-dimensional matrices.</a> Curr Opin Cell Biol. 14, 633-639 (2002).</li><li>Nelson, C. M., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+extracellular+matrix,+scaffolds,+and+signaling:+tissue+architecture+regulates+development,+homeostasis,+and+cancer.">Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer.</a> Annu Rev Cell Dev Biol. 22, 287-309 (2006).</li><li>Davis, G. S., Phillips, H. M., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-layer+surface+tensions+and+&amp;quot;tissue+affinities&amp;quot;+in+Rana+pipiens+gastrulae:+quantitative+measurements.">Germ-layer surface tensions and &amp;quot;tissue affinities&amp;quot; in Rana pipiens gastrulae: quantitative measurements.</a> Dev Biol. 192, 630-644 (1997).</li><li>Schotz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+differences+in+tissue+surface+tension+influence+zebrafish+germ+layer+positioning.">Quantitative differences in tissue surface tension influence zebrafish germ layer positioning.</a> HFSP J. 2, 42-56 (2008).</li><li>Jia, D., Dajusta, D., Foty, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+surface+tensions+guide+in+vitro+self-assembly+of+rodent+pancreatic+islet+cells.">Tissue surface tensions guide in vitro self-assembly of rodent pancreatic islet cells.</a> Dev Dyn. 236, 2039-2049 (2007).</li><li>Foty, R. A., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differential+adhesion+hypothesis:+a+direct+evaluation.">The differential adhesion hypothesis: a direct evaluation.</a> Dev Biol. 278, 255-263 (2005).</li><li>Li, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+casein+kinase+2+subunits+in+Chinese+hamster+ovary+and+3T3+L1+cells+provides+information+on+the+role+of+the+enzyme+in+cell+proliferation+and+the+cell+cycle.">Expression of the casein kinase 2 subunits in Chinese hamster ovary and 3T3 L1 cells provides information on the role of the enzyme in cell proliferation and the cell cycle.</a> J Biol Chem. 274, 32988-32996 (1999).</li><li>Foty, R. A., Pfleger, C. M., Forgacs, G., Steinberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+tensions+of+embryonic+tissues+predict+their+mutual+envelopment+behavior.">Surface tensions of embryonic tissues predict their mutual envelopment behavior.</a> Development. 122, 1611-1620 (1996).</li><li>Wendt, D., Riboldi, S. A., Cioffi, M., Martin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+and+bottlenecks+of+bioreactors+in+3D+cell+culture+and+tissue+manufacturing.">Potential and bottlenecks of bioreactors in 3D cell culture and tissue manufacturing.</a> Adv Mater. 21, 3352-3367 (2009).</li><li>Berry, D. P., Harding, K. G., Stanton, M. R., Jasani, B., Ehrlich, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+wound+contraction:+collagen+organization,+fibroblasts,+and+myofibroblasts.">Human wound contraction: collagen organization, fibroblasts, and myofibroblasts.</a> Plast Reconstr Surg. 102, 124-131 (1998).</li><li>Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+biology+in+three-dimensional+collagen+matrices.">Fibroblast biology in three-dimensional collagen matrices.</a> Trends Cell Biol. 13, 264-269 (2003).</li><li>Wang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+interactions+between+beta1-integrin+and+epidermal+growth+factor+receptor+in+three-dimensional+basement+membrane+breast+cultures:+a+different+perspective+in+epithelial+biology.">Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology.</a> Proc Natl Acad Sci U S A. 95, 14821-14826 (1998).</li><li>Weaver, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+the+malignant+phenotype+of+human+breast+cells+in+three-dimensional+culture+and+in+vivo+by+integrin+blocking+antibodies.">Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.</a> J Cell Biol. 137, 231-245 (1997).</li><li>Boudreau, N., Werb, Z., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+apoptosis+by+basement+membrane+requires+three-+dimensional+tissue+organization+and+withdrawal+from+the+cell+cycle.">Suppression of apoptosis by basement membrane requires three- dimensional tissue organization and withdrawal from the cell cycle.</a> Proc Natl Acad Sci U S A. 93, 3509-3513 (1996).</li><li>Croix, B., Rak, J. W., Kapitain, S., Sheehan, C., Graham, C. H., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+by+hyaluronidase+of+adhesion-dependent+multicellular+drug+resistance+in+mammary+carcinoma+cells.">Reversal by hyaluronidase of adhesion-dependent multicellular drug resistance in mammary carcinoma cells.</a> J. Nat. Cancer Inst. 88, 1285-1296 (1996).</li><li>Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+structure,+nuclear+organization,+and+gene+expression+in+normal+and+malignant+breast.">Tissue structure, nuclear organization, and gene expression in normal and malignant breast.</a> Cancer Res. 59, 1757-1763 (1999).</li><li>Marrero, B., Messina, J. L., Heller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+tumor+spheroid+in+a+microgravity+environment+as+a+3D+model+of+melanoma.">Generation of a tumor spheroid in a microgravity environment as a 3D model of melanoma.</a> In Vitro Cell Dev Biol Anim. 45, 523-534 (2009).</li><li>Napolitano, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffold-free+three-dimensional+cell+culture+utilizing+micromolded+nonadhesive+hydrogels.">Scaffold-free three-dimensional cell culture utilizing micromolded nonadhesive hydrogels.</a> Biotechniques. 43, 494-496 (2007).</li><li>Tibbitt, M. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+as+extracellular+matrix+mimics+for+3D+cell+culture.">Hydrogels as extracellular matrix mimics for 3D cell culture.</a> Biotechnol Bioeng. 103, 655-663 (2009).</li></ol>

利害の衝突は宣言されません。

研究では、三次元コンテキスト（3D）での培養細胞が異なる細胞の形態とリジッド2次元（2D）文化系9と比較してシグナル伝達をもたらすことが示されている。例えば、線維芽細胞に移入コラーゲンゲルは、3Dでの線維芽細胞の形態は、2D 10,11で観察されたものとはかなり異なっていることを示しています。同様に、三次元培養では、乳腺上皮細胞の組織特異的分化を誘導する?...

<ul><li>自動セルカウンター（バイオラッドTC10） </li><li> CO 2切妻のカバーモデル3540（ラボライン）で水浴を揺るがす</li></ul>

a simple hanging drop cell culture protocol for generation of 3d spheroids

細胞間の凝集と細胞 - 基質接着の研究は歴史的に硬質基材への付着性単層培養で行われている。組織内の細胞は、しかし、一般的に細胞は多くの近い隣人とし、細胞外マトリックス成分との親密な接続を確立した最密充填組織塊内に収められています。したがって、化学的環境と3次元組織内の細胞が経験する物理的な力は、単層培養で増殖させた細胞で経験したものよりも根本的に異なっています。これは、著しく細胞形態及びシグナル伝達に影響を与えることが示されている。いくつかのメソッドは、コラーゲンゲル1または生体材料足場2のセルのカプセル化を含む3次元細胞培養を生成するために考案されている。このような方法は、便利な一方、正常組織に見られる親密な直接細胞間接着のアーキテクチャを要約することはありません。単一の細胞が疎なECM製品の3次元網目構造内に分散されているのではなく、彼らがより密接に近似する培養システム。ここで、我々は、彼らは、細胞同士や細胞外マトリックス成分と直接接触している真の3D回転楕円体を形成するまで細胞がドロップ文化をハング内に配置し、生理的条件下でインキュベートされる、単純な方法を説明します。メソッドは特殊な装置を必要とせず、細胞間または細胞- ECM相互作用への影響を解明することに関心があると思われる非常に少量の任意の生物学的因子の付加を含むように適合させることができます。メソッドは、セル間の空間関係を指定するには、細胞間や細胞 - ECM間の相互作用の役割を解明するために共培養つ（以上）の異なる細胞集団にも使用できます。細胞間の凝集と細胞- ECMの接着は胚発生の研究の基礎、悪性浸潤における腫瘍間質細胞の相互作用、創傷治癒、及び組織工学への応用のためです。この簡単な方法は、生体力学的特性の測定のためのまたは生理学的に関連するモデルの分子的および生化学的解析のための細胞凝集のような組織を生成する手段を提供します。

Watch this Scientific Journal Video about A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids at JoVE.com

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

我々は、細胞 - 細胞相互作用の違いを定量化するために三次元組織のような回転楕円体とそれらの潜在的なアプリケーションを生成する単純な、急速な方法を説明します。

3D楕円体の生成のための簡単​​なハンギングドロップ細胞培養プロトコル

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

a-simple-hanging-drop-cell-culture-protocol-for-generation-3d

Research

JoVE Journal

Bioengineering

56.6K ビュー.  UMDNJ-Robert Wood Johnson Medical School. 我々は、細胞 - 細胞相互作用の違いを定量化するために三次元組織のような回転楕円体とそれらの潜在的なアプリケーションを生成する単純な、急速な方法を説明します。

ビデオ: A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

幹細胞培養アプリケーションのための3Dフィブリン足場の調製

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

生物工学

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

ステップ成長PEG-ペプチドハイドロゲルからβ細胞のスフェロイドの発生と復旧

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

関節軟骨細胞培養軟骨の生成のための3Dハイドロゲルの足場

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3D細胞培養のための肺細胞外マトリックスから調整可能なヒドロゲル

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

リアルタイム 3 D 単一粒子追跡のためのプロトコル

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3 D ダイナミック文化システムの多層間葉系幹細胞シートの構築

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

マトリックス細胞代謝クロストークを研究するためのヒト3D細胞外マトリックス-脂肪細胞培養モデル

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

3Dバイオプリンティングのための脂肪由来幹細胞スフェロイドの大規模自動生産

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

三次元細胞培養基材のUVフォトパターニングによるマルチキュー細胞微小環境の生成

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

ヒト誘起多能性幹細胞由来心臓スフェロイドの作製、ハイスループットスクリーニング、バイオバンキング

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

3D楕円体の生成のための簡単​​なハンギングドロップ細胞培養プロトコル

3D楕円体の生成のための簡単なハンギングドロップ細胞培養プロトコル