Name: 著者スポットライト: 3D オルガノイドモデルによる腱組織工学の進歩
Uploaded: 2024-06-21T13:00:00
Description: Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

D.D.とS.M.-D.BMBF助成金「CellWiTaL:医薬品研究のための再現性のある細胞システム-三次元細胞構造における非常に特異的な単一細胞の転写層フリーレーザー印刷」提案番号13N15874に感謝します。D.D.とV.R.A.は、EU MSCA-COFUND GRANT OSTASKILLSの「次世代の変形性関節症研究のホリスティックトレーニング」GA Nr.101034412を認めています。すべての著者は、技術支援を提供してくれたベアテ・ガイヤー夫人に感謝します。

注:すべてのステップは、無菌技術を使用して実行する必要があります。
1. NHDFの培養と事前拡大
<ol><li>成人の凍結保存された正常なヒト皮膚線維芽細胞(NHDF、1 x 106 細胞)を含むクライオバイアルを、ほぼ解凍するまで37°Cで急速に解凍します。</li>
	<li>1%ペニシリン/ストレプトマイシン(ペン/連鎖球菌)を補充した1mLの予熱した線維芽細胞増殖培地2(基?...

腱の研究とエンジニアリングのための3ステップオルガノイドモデル

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+organoids:+model+systems+for+human+biology+and+medicine.">Human organoids: model systems for human biology and medicine.</a> Nat Rev Mol Cell Biol. 21 (10), 571-584 (2020).</li><li>Hsieh, C. F., 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=In+vitro+comparison+of+2D-cell+culture+and+3D-cell+sheets+of+scleraxis-programmed+bone+marrow+derived+mesenchymal+stem+cells+to+primary+tendon+stem/progenitor+cells+for+tendon+repair.">In vitro comparison of 2D-cell culture and 3D-cell sheets of scleraxis-programmed bone marrow derived mesenchymal stem cells to primary tendon stem/progenitor cells for tendon repair.</a> Int J Mol Sci. 19 (8), 2272 (2018).</li><li>Chu, J., Lu, M., Pfeifer, C. G., Alt, V., Docheva, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuilding+tendons:+A+concise+review+on+the+potential+of+dermal+fibroblasts.">Rebuilding tendons: A concise review on the potential of dermal fibroblasts.</a> Cells. 9 (9), 2047 (2020).</li><li>Gaspar, D., Spanoudes, K., Holladay, C., Pandit, A., Zeugolis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+cell-based+therapies+for+tendon+repair.">Progress in cell-based therapies for tendon repair.</a> Adv Drug Deliv Rev. 84, 240-256 (2015).</li><li>Sacco, A. M., 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=Diversity+of+dermal+fibroblasts+as+major+determinant+of+variability+in+cell+reprogramming.">Diversity of dermal fibroblasts as major determinant of variability in cell reprogramming.</a> J Cell Mol Med. 23 (6), 4256-4268 (2019).</li><li>Wang, W., 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=Induction+of+predominant+tenogenic+phenotype+in+human+dermal+fibroblasts+via+synergistic+effect+of+TGF-&#946;+and+elongated+cell+shape.">Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-&#946; and elongated cell shape.</a> Am J Physiol Cell Physiol. 310 (5), C357-C372 (2016).</li><li>Pryce, B. A., Watson, S. S., Murchison, N. D., Staverosky, J. 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この研究で実証された結果は、T/L 組織を研究するための NHDF 3D オルガノイド モデルの確立と特性評価に関する貴重な洞察を提供します。3ステップのプロトコルにより、T/Lニッチの典型的な特徴を示す3D棒状オルガノイドが形成されました。このモデルは、以前にKroner-Weigl et al.2023 :7 で報告されており、ここで非常に詳細に実証されています。
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腱と靭帯(T / L)は、体に不可欠なサポートと安定性を提供する筋骨格系の重要な構成要素です。これらの結合組織は、その重要な役割にもかかわらず、変性や損傷を起こしやすく、痛みや運動障害を引き起こします1。さらに、それらの限られた血液供給と遅い治癒能力は慢性的な怪我につながる可能性がありますが、老化、反復運動、不適切なリハビリテーションなどの要因は、変性や怪我のリスクをさらに高めます2。安静、理学療法、外科的介入などの従来の治療法では、T/Lの構造と機能を完全に回復させることはできません。過去数年間、研究者は T/L 障害の効果的な治療法を模索するために、T/L の複雑な性質をよりよく理解しようと努めてきました 3,4,5。T/Lは、主にI型コラーゲン線維とプロテオグリカンで構成される、階層的に組織化された細胞外マトリックス(ECM)優位の構造によって区別されますが、これはin vitroで再現することが困難な特徴です6。従来の2次元(2D)細胞培養モデルは、T/L組織の特徴的な3次元(3D)組織を捉えることができず、翻訳の可能性を制限し、T/L再生の分野における革新的な進歩を妨げていました。
近年、3Dオルガノイドモデルの開発により、基礎研究や様々な組織タイプのスキャフォールドフリー組織工学を前進させる新たな可能性がもたらされました7,8,9,10,11,12,13。例えば、筋腱接合部を調査するために、Larkin et al. 2006は、ラット尾腱に由来する自己組織化腱セグメントとともに3D骨格筋構造を開発した10。さらに、Schiele et al. 2013は、マイクロマシニングされたフィブロネクチン被覆成長チャネルを使用することにより、ヒト真皮線維芽細胞の自己組織化を指示し、3D足場の助けを借りずに細胞線維を形成することを示し、胚性腱発生の重要な形質を捉えることができるアプローチである11。Florida et al. 2016による研究では、骨髄間質細胞が最初に骨と靭帯の系統に拡大され、次に自己組織化された単層細胞シートを生成するために使用され、次に実装されて、靭帯再生の理解を深めることを目的としたモデルである、天然の前十字靭帯を模倣した多相性の骨-靭帯-骨構造を作成しました12.腱のメカノトランスダクションプロセスを解明するために、Mubyana et al. 2018は、単一の腱繊維を作成し、機械的負荷プロトコル13にかける足場のない方法を利用しました。オルガノイドは、組織の本来の構造、微小環境、機能性を模倣した自己組織化された3D構造です。3Dオルガノイド培養は、組織や臓器の生物学、病態生理学を研究するための、より生理学的に適切なモデルを提供します。このようなモデルは、異なる幹細胞/前駆細胞型の組織特異的分化を誘導するためにも使用できます14,15。したがって、T/L生物学および組織工学の分野で3Dオルガノイドモデルを実装することは、非常に魅力的なアプローチになります9,16。オルガノイド集合体に代替の細胞源を実装し、テノイド分化に向けて刺激することができます。この研究でデモンストレーションに使用された関連する細胞タイプの1つは、真皮線維芽細胞7,17,18です。これらの細胞は、骨髄穿刺や脂肪吸引に比べて侵襲性が低く、増殖能力が高いため、かなり迅速に大量に増殖できる皮膚生検手順によって簡単にアクセスできます。対照的に、T/L常在性線維芽細胞などのより特殊な細胞タイプは、単離と増殖がより困難です。したがって、真皮線維芽細胞は、人工多能性胚性幹細胞に向けた細胞リプログラミング技術の出発点としても使用された19。T/Lの形成と維持を含むさまざまな細胞プロセスの主要な調節因子として作用することが報告されているトランスフォーミング成長因子-β3(TGFß3)などの特定の3D培養条件とシグナル伝達の手がかりに真皮線維芽細胞をさらすと、腱特異的遺伝子の発現とT/L典型的なECMの沈着につながるin vitroのテノジェニック分化を増強できます20、21.
ここでは、市販の正常な成人ヒト皮膚線維芽細胞(NHDF)を細胞源として使用して、以前に確立および実装された3ステップ(2D拡張、2D刺激、および3D成熟)オルガノイドプロトコルについて説明し、実証し、 in vitro 腱形成を研究するための貴重なモデルを提供します7。このモデルは in vivo T/L組織と同等ではないという事実にもかかわらず、細胞分化メカニズムの調査、 in vitroでのT/L病態生理学の模倣、およびT/L個別化医療および薬物スクリーニングプラットフォームの確立に使用できる、より生理学的に関連性の高いシステムを提供します。さらに、将来的には、3Dオルガノイドが、天然のT/L組織の寸法や構造的および生物物理学的特性によく似た、スケールアップされた機械的に堅牢な構造の開発に利用できるだけでなく、さらなる最適化によって、足場のないT/Lエンジニアリングに適しているかどうかを評価することができます。

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			<td>Ascorbic acid&#160;&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;&#160;</td>
			<td>A8960</td>
			<td></td>
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			<td>10 cm adherent cell culture dish</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430167</td>
			<td></td>
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			<td>10 cm non-adherent petri dish&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen,Germany&#160;</td>
			<td>CLS430591</td>
			<td></td>
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			<td>Cryo-medium</td>
			<td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands&#160;&#160;</td>
			<td>4583</td>
			<td></td>
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			<td>Cryomold standard&#160;</td>
			<td>Tissue-Tek, Sakura Finetek, Alphen aan den Rijn, Netherlands</td>
			<td>4557</td>
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			<td>D(+)-Sucrose&#160;</td>
			<td>AppliChem Avantor VWR International GmbH, Darmstadt, Germany</td>
			<td>A2211</td>
			<td></td>
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			<td>DMEM high glucose medium&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>DMEM-HA</td>
			<td></td>
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			<td>DMEM low glucose</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>DMEM-LPXA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>&#160;Anprotec, Bruckberg, Germany&#160;</td>
			<td>AC-SM-0027</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast growth medium 2&#160;</td>
			<td>PromoCell, Heidelberg, Germany&#160;&#160;</td>
			<td>C-23020</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope with high resolution camera</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td>Zeiss Axio Observer with&#160; Axiocam 506</td>
		</tr>
		<tr>
			<td>MEM amino acids&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;&#160;</td>
			<td>NEAA-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal pins&#160;</td>
			<td>EntoSphinx, Pardubice, Czech Republic&#160;</td>
			<td>04.31</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal human dermal fibroblasts&#160;</td>
			<td>&#160;PromoCell, Heidelberg, Germany&#160;</td>
			<td>C-12302</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>&#160;AppliChem, Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>A3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin&#160;</td>
			<td>Gibco, Thermo Fisher Scientific, Darmstadt, Germany</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer saline&#160;</td>
			<td>Sigma-Aldrich, Taufkirchen, Germany&#160;</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#223;3&#160;</td>
			<td>R&#38;D Systems, Wiesbaden, Germany&#160;</td>
			<td>&#160;8420-B3</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0,05% DPBS&#160;</td>
			<td>Capricorn Scientific, Ebsdorfergrund, Germany&#160;</td>
			<td>&#160;TRY-1B</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstration of self-assembled cell sheet culture and manual generation of a 3d tendon/ligament-like organoid by using human dermal fibroblasts

腱と靭帯(T / L)は、筋骨格系を結合する強力な階層的に組織化された構造です。これらの組織は、厳密に配置されたコラーゲンI型に富む細胞外マトリックス(ECM)とT/L系統細胞を主に平行に並べています。怪我の後、T/Lはリハビリテーションに長い時間を必要とし、故障のリスクが高く、多くの場合、満足のいく修復結果が得られません。近年のT/L生物学研究の進歩にもかかわらず、残された課題の1つは、T/L分野には、 in vitroでT/L形成プロセスを再現できる標準化された分化プロトコルがまだないことです。例えば、間葉系前駆細胞の骨と脂肪の分化には、標準的な2次元(2D)細胞培養と特異的刺激培地の添加のみが必要です。軟骨への分化には、3次元(3D)ペレット培養とTGFβの補給が必要です。しかし、腱への細胞分化には、非常に整然とした3D培養モデルが必要であり、理想的には動的な機械的刺激にもさらされる必要があります。私たちは、自己組織化された細胞シートから3次元の棒状構造を形成する3段階(膨張、刺激、成熟)のオルガノイドモデルを確立し、ECM、オートクライン、パラクリン因子を持つ自然な微小環境を作り出しています。これらの棒状オルガノイドは、豊富なECM内に多層の細胞構造を持ち、静的な機械的ひずみにさらされても非常に簡単に扱うことができます。ここでは、市販の真皮線維芽細胞を用いて3ステップのプロトコールを実証しました。この細胞タイプが、強固でECMに富んだオルガノイドを形成することを示すことができました。記載された手順は、培地に関してさらに最適化され、動的軸方向の機械的刺激に向けて最適化され得る。同様に、代替細胞源は、T/Lオルガノイドを形成し、T/L分化を起こす可能性についてテストすることができます。要するに、確立された3D T/Lオルガノイドアプローチは、腱の基礎研究のモデルとして、さらには足場のないT/Lエンジニアリングにも使用できます。

Tendons and ligaments (T/L) are vital components of the musculoskeletal system that provide essential support and stability to the body. Despite their critical role, these connective tissues are prone to degeneration and injury, causing pain and impairment of mobility1. Moreover, their limited blood supply and slow healing capacity can lead to chronic injuries, whereas factors such as aging, repetitive motion, and improper rehabilitation further increase the risk of degeneration and injury2. Conventional treatments, such as rest, physical therapy, and surgical interventions, are unable to fully restore the T/L structure and function. Over the past few years, researchers have strived to better understand the intricate nature of T/L in order to seek effective treatments for T/L disorders3,4,5. T/L are distinguished by a hierarchically organized, extracellular matrix (ECM)-dominated structure, composed primarily of type I collagen fibers and proteoglycans, a feature that is difficult to be replicated in vitro6. Traditional two-dimensional (2D) cell culture models fail to capture the characteristic three-dimensional (3D) organization of T/L tissues, limiting their translational potential as well as hindering the innovative progress in the field of T/L regeneration.
Recently, the development of 3D organoid models has offered new possibilities for advancing basic research and scaffolds-free tissue engineering of various tissue types7,8,9,10,11,12,13. For example, to investigate myotendinous junction, Larkin et al. 2006 developed 3D skeletal muscle constructs together with self-organized tendon segments derived from rat tail tendon10. Moreover, Schiele et al. 2013, by using micromachined fibronectin-coated growth channels, directed the self-assembly of human dermal fibroblasts to form cellular fibers without the assistance of 3D scaffold, an approach that can capture key traits of embryonic tendon development11. In the study by Florida et al. 2016, bone marrow stromal cells were first expanded into bone and ligament lineages, next used to generate self-assembled monolayer cell sheets, which were then implemented to create a multiphasic bone-ligament-bone construct mimicking the native anterior cruciate ligament, a model aiming at improved understanding of ligament regeneration12. To elucidate tendon mechanotransduction processes, Mubyana et al. 2018 utilized a scaffold-free methodology by which single tendon fibers were created and subjected to mechanical loading protocol13. Organoids are self-organized 3D structures that mimic the native architecture, microenvironment, and functionality of tissues. 3D organoid cultures provide a more physiologically relevant model for studying tissue and organ biology as well as pathophysiology. Such models can also be used to induce tissue-specific differentiation of different stem/progenitor cell types14,15. Hence, implementing 3D organoid models in the field of T/L biology and tissue engineering becomes a very attractive approach9,16. Alternative cell sources can be implemented for the organoid assembly and stimulated toward tenogenic differentiation. One relevant cell type used for demonstration in this study is dermal fibroblasts7,17,18. These cells are easily accessible through a skin biopsy procedure, which is less invasive compared to bone marrow puncture or liposuction and can be multiplied fairly quickly to large numbers due to their good proliferative capacity. In contrast, more specialized cell types, such as T/L-resident fibroblasts, are more challenging to isolate and expand. Therefore, dermal fibroblasts were also used as a starting point for cell reprogramming technologies towards induced pluripotent embryonic stem cells19. Subjecting dermal fibroblasts to specific 3D culture conditions and signaling cues, such as transforming growth factor-beta 3 (TGF&#223;3), which has been reported to act as a key regulator of various cellular processes, including the formation and maintenance of T/L, can potentiate their in vitro tenogenic differentiation leading to the expression of tendon-specific genes and the deposition of T/L-typical ECM20,21.
Here, we describe and demonstrate a previously established and implemented 3-step (2D expansion, 2D stimulation, and 3D maturation) organoid protocol using commercially available normal adult human dermal fibroblasts (NHDFs) as a cell source, offering a valuable model for studying in vitro tenogenesis7. Despite the fact that this model is not equivalent to in vivo T/L tissue, it still provides a more physiologically relevant system that can be used for investigating cellular differentiation mechanisms, mimicking T/L pathophysiology in vitro, and establishing T/L personalized medicine and drug screening platforms. Moreover, in the future, studies can evaluate whether the 3D organoids are suitable for scaffold-free T/L engineering by further optimization as well as utilizable for the development of scaled-up mechanically robust constructs that closely resemble the dimensions and structural and biophysical properties of native T/L tissues.

著者スポットライト: 3D オルガノイドモデルによる腱組織工学の進歩

ヒト真皮線維芽細胞を用いた自己組織化細胞シート培養の実証と3次元腱・靭帯様オルガノイドの手動生成

We demonstrate a previous established and implement three-step organoid protocol using commercially available, normal human dermal fibroblasts as a cell source. The 3D organoid model holds several advantage for tissue engineering and regenerative medicine application. It mimics closely the cellular composition and organization as well as cell-to-cell and cell-to-matrix interactions of tendon tissues.The established 3D tendon-ligament organoid approach can be used as a model for tendon basic and applied research in the following areas. In vitro tenogenesis, disease mechanism, drug testing, and scaffold-free tendon tissue engineering.

3D T/Lオルガノイドモデルは、市販のNHDFを実施することで以前に確立され、ここで実証されました(n = 3、ドナーあたり3つのオルガノイド、NHDFは継代5〜8で使用されました)。モデルのワークフローを 図 1 にまとめます。 図2 は、T-75フラスコでの膨張前(図2A)と、10cm細胞培養皿での2D増殖ステップでの培養の開始時と5日間の培養...

ここでは、腱の基礎研究に有望なツールと、腱組織工学のための潜在的な足場のない方法を提供する3ステップオルガノイドモデル(2次元[2D]拡張、2D刺激、3次元[3D]成熟)を実証します。

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

私たちは、以前に確立された3段階のオルガノイドプロトコルを実証し、市販の正常なヒト真皮線維芽細胞を細胞源として使用します。3Dオルガノイドモデルは、組織工学や再生医療への応用においていくつかの利点を持っています。これは、細胞の組成と組織、および腱組織の細胞間および細胞とマトリックスの相互作用を厳密に模倣しています。確立された3D腱靭帯オルガノイドアプローチは、以下の分野で腱の基礎研究と応用研究のモデルとして使用できます。in vitro腱形成、疾患メカニズム、薬物試験、および足場のない腱組織工学。

demonstration-self-assembled-cell-sheet-culture-manual-generation-3d

Research

JoVE Journal

Bioengineering

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly arranged collagen type&#160;I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGF&#223; is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Herein, we demonstrate a three-step organoid model (two-dimensional [2D] expansion, 2D stimulation, three-dimensional [3D] maturation) offering a promising tool for tendon fundamental research and a potential scaffold-free method for tendon tissue engineering.