この研究は、NIH 1R01HL140239-01によってAMCに資金提供されました。ドレクセル大学の細胞イメージングセンターに感謝します。

著者らは開示するものは何もない。

このプロトコルは、3Dアーキテクチャにおいても内皮細胞との共培養のための3Dアーキテクチャにおけるバイオプリントスフェロイドの初のプロトコルです。重要なプロトコルステップには、乳房上皮スフェロイドおよびHUVECネットワークの初期形成が含まれる。乳房上皮スフェロイドはマトリックス溶液から容易に破壊されるため、授乳中には細心の注意が必要です。同様に、乳房上皮スフ?...

3D in vitro血管化腫瘍モデルは、がんの成長と転移の機械化研究に不可欠なツールです。特に乳癌の場合、マトリゲルで培養された乳房上皮細胞は、生体内乳腺アシヌスアーキテクチャ1、2、3、4、5、6、7、8に似た偏光スフェロイドに整理する。3D乳房上皮細胞培養も細胞機能に影響を与え、表皮成長因子(EGF)受容体変調8、9の違いを示す3D培養で、オンコジーン機能, ErbB210を含む;成長とアポトーシスシグナル11、12;と化学療法抵抗13,14.血管内皮細胞は、従来の2D培養15、16、17、18との3Dと同様に環境刺激に対して異なる反応を示す。しかし、血管内皮および乳房上皮相互作用の理解の多くは、2D培養に条件付き培地またはトランスウェル挿入物を用い、または2つの細胞タイプが物理的に19、20、21、22、23に分離された3Dモデルから来ている。これらの共培養モデルは、3D培養と細胞-細胞接触の両方が血管内皮に重要であるので、限られた生理学的洞察力を提供する – 乳房上皮細胞相互作用24,25,26.
3Dがんモデルは、ヒドロゲル内または設計された足場5、27、28、29の中で、吊り下げスフェロイド形成、バイオプリンティング、磁気アセンブリ、培養を含む様々な技術を用いて製造されている。最近では、3D腫瘍モデルが、それぞれの3D構造に配置された複数の細胞タイプで作成されました。腫瘍オンチッププラットフォームの一例では、癌、内皮細胞、および間質細胞をマトリックスに混合し、次いでポリジメチルシロキサン(PDMS)装置の中の3つの中央組織室に注入した。組織室は、動脈と静脈を表す2つの外側のチャネルで接していた。培養の5〜7日後、内皮細胞は血管系の近くに小さな腫瘍を形成するために増殖した微小血管網と癌細胞を形成した。このプラットフォームは、その後、スクリーニング薬と薬物の組み合わせ30に使用されました。特定の機械的刺激(例えば、肺の機械的緊張)31,32を有する転移および癌タイプを研究するために、追加の腫瘍オンチッププラットフォームが作成された。しかし、これらのプラットフォームは、一般的にそれぞれの3D構造に血管系と癌の両方を含みはありません。
バイオファブリケーションは、細胞の位置を厳密に空間制御できるため、3D in vitro血管化腫瘍モデルを進める上で大きな約束を示しています。過去10年間のバイオプリンティングの成長にもかかわらず、腫瘍33、34に特に焦点を当てた研究はほとんどありません。ある例では、ゼラチン/アルギン酸/フィブリノーゲンヒドロゲル中のHeLa細胞の3Dプリンティングを使用して、インビトロ子宮頸癌モデルを作成した。腫瘍細胞を個々の細胞としてバイオプリントし、次いでスフェロイドを形成させ、これはより高い増殖速度、マトリックスメタロプロテイナーゼ発現の増加、および2D培養35における細胞よりも高い化学抵抗性を示した。これらの研究では、他の多くの36,37と同様に、解ciaciaされた細胞懸濁液をバイオプリントし、次に細胞培養物に必要な機械的および生化学的手掛かりを与えて細胞が3D構造を形成することを可能にした。しかし、細胞自己集合は数日または数週間かかる場合があり、複雑な空間的および時間的環境的手掛かりを必要とすることがあり、または2つの細胞タイプが共同培養される場合には起こらない場合がある。例えば、2D共培養において内皮細胞における細胞死を誘導した乳房上皮細胞、および解約乳房上皮細胞は、アルギン酸/ゼラチンヒドロゲル38にバイオプリントした際に3Dスフェロイドを形成しなかった。解体乳房上皮または癌細胞は、円形PDMS型に包絡された場合にのみ、アルギン酸塩ベースのバイオインクでスフェロイドを形成した。他の場合には、スフェロイドは、超低添付着円状ウェルプレート中に懸濁液滴を使用して形成し、次いでアルギン酸系バイオインク39,40に混合した。
このプロトコルでは、代替3D組織バイオマニュファクチャリング法について説明します。解約細胞を播種してこれらの細胞が3D構造を形成するのを待つのではなく、血管管ネットワーク上に3D腫瘍回転楕円体を作成してバイオプリントし、ほぼ即座に使用できる腫瘍共培養モデルを作成する方法を説明する。腫瘍スフェロイドは、インビトロで成長させるか、またはヒト組織(オルガノイド)に由来する。同様に、血管管は、微小血管断片の脂肪組織に由来することができるか、または成長させることができる。バイオインクは、生物学的に不活性なアルギン酸から、生物学的に活性の高いマトリゲル41まで及ぶ。この3D腫瘍共培養モデルは、様々な細胞構造およびバイオインクを用いて作成することができるので、複数の細胞型、細胞外マトリックス、およびケモカイン勾配15、42を組み込むことができる。現在の製剤では、内皮ネットワークを浸透させることはできませんが、将来の反復は、この方法をマイクロ流体またはオンチップシステムと統合することができます。内皮ネットワーク上のバイオプリンティング3D乳房上皮スフェロイドは、薬物検査および個別の精密医療27のためのヒト乳房モデルの迅速なバイオファブリケーションを可能にする。

<table><tbody><tr><td>37&amp;deg;C incubator, 5% CO2 and 95% humidity</td><td>Sanyo</td><td>MCO-20AIC</td><td>Cell incubation</td></tr><tr><td>3D Bio printer</td><td>custom-made</td><td>None</td><td>Used for bioprinting</td></tr><tr><td>8-well chamber slides</td><td>VWR, Radnor, PA</td><td>53106-306</td><td>for seeding spheroids</td></tr><tr><td>25-gauge needle</td><td>Sigma, St. Louis, MO</td><td>Z192406-100EA</td><td>bioprinting syringe needle</td></tr><tr><td>Absolute ethanol (200 proof )</td><td>Sigma, St.Louis, MO</td><td>E7023-500ML</td><td>reconsitution of media components</td></tr><tr><td>Affinipure F(ab&amp;prime;)2 fragment goat anti-mouse IgG</td><td>Jackson ImmunoResearch, West Grove, PA</td><td>115006020</td><td>secondary block - Immunofluorescence</td></tr><tr><td>Alexa Fluor 488 (1:200)</td><td>Thermo Fisher, Waltham, MA</td><td>A-11006</td><td>Seconday antibody-Immunofluorescence</td></tr><tr><td>Bovine insulin</td><td>Sigma, St.Louis, MO</td><td>I-035-0.5ML</td><td>MCF10A Media additive</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma, St.Louis, MO</td><td>A2153-500G</td><td>Blocking agent -Immunofluorescence</td></tr><tr><td>Falcon 70 &amp;micro;m Cell Strainer</td><td>Corning, Corning, NY</td><td>352350</td><td>Remove large or clustered spheroids</td></tr><tr><td>CellTracker&amp;trade; Red CMTPX Dye</td><td>Thermo Fisher, Waltham, MA</td><td>C34552</td><td>pre-stain for HUVEC tubes</td></tr><tr><td>Compact Centrifuge</td><td>Hermle- Labnet, Edison ,NJ</td><td>Z206A</td><td>For cell centrifugations</td></tr><tr><td>Cholera Toxin</td><td>Sigma, St.Louis, MO</td><td>C8052-.5MG</td><td>MCF10A Media additive</td></tr><tr><td>Conical tubes 15 mL</td><td>VWR, Radnor, PA</td><td>62406-200</td><td>Collecting and resuspending cells</td></tr><tr><td>Countess II-FL Cell counter</td><td>Thermo Fisher, Waltham, MA</td><td>AMQAF1000</td><td>counting cells</td></tr><tr><td>Glass pipettes (10 mL)</td><td>VWR, Radnor, PA</td><td>76184-746</td><td>cell resuspension</td></tr><tr><td>DMEM F:12</td><td>Thermo Fisher, Waltham, MA</td><td>11320033</td><td>MCF10A basal media</td></tr><tr><td>DMEM 1X</td><td>VWR, Radnor, PA</td><td>10-014-CV</td><td>MDA-MB-231 basal media</td></tr><tr><td>Endothelial Basal Medium-2 (EBM-2)</td><td>Lonza, Durham, NC</td><td>CC-3156</td><td>HUVEC basal media</td></tr><tr><td>Endothelial Growth Medium-2 (EGM-2)</td><td>Lonza, Durham, NC</td><td>CC-3162</td><td>Accompanied with a Bulletkit (containing growth factors)</td></tr><tr><td>Alexa Fluor&amp;trade; 488 Phalloidin</td><td>Thermo Fisher, Waltham, MA</td><td></td><td>Labelling MDA-MB-231 spheroids</td></tr><tr><td>Fetal Bovine serum</td><td>Cytiva, Logan, UT</td><td>SH30071.03</td><td>HUVEC/MDA-MB-231 media additive</td></tr><tr><td>Goat serum</td><td>Thermo Fisher, Waltham, MA</td><td>16210064</td><td>Immunofluorescence labelling component</td></tr><tr><td>Glycine</td><td>Sigma, St.Louis, MO</td><td>G8898-500G</td><td>immunofluorescence buffer component</td></tr><tr><td>Hoescht 33342</td><td>Thermo Fisher, Waltham, MA</td><td>62249</td><td>Nuclei stain immunofluorescence</td></tr><tr><td>Horse Serum</td><td>Thermo Fisher, Waltham, MA</td><td>16050130</td><td>MCF10A Media additive</td></tr><tr><td>Hydrocortisone</td><td>Sigma, St.Louis, MO</td><td>H0888-5G</td><td>MCF10A Media additive</td></tr><tr><td>Human Umblical Vein Endothelial cells (HUVECs)</td><td>Cell applications, San Diego , CA</td><td>200-05f</td><td>Endothelial cell lines</td></tr><tr><td>Integrin &amp;alpha;6</td><td>Millipore, Billerica, MA</td><td>MAB1378</td><td>Immunofluorescence spheroid labelling component</td></tr><tr><td>Live Dead assay</td><td>Thermo Fisher, Waltham, MA</td><td>L3224</td><td>Live and dead cell stain assay for cell viability</td></tr><tr><td>LSM 700 Confocal microscope</td><td>Zeiss, Thornwood, NY</td><td></td><td>Used to visualize cells</td></tr><tr><td>Matrigel - growth factor reduced 10 mg/ml</td><td>VWR, Radnor, PA</td><td>354230</td><td>Spheroid formation</td></tr><tr><td>MCF10A cells</td><td>ATCC</td><td>CRL-10317</td><td>Breast cell line</td></tr><tr><td>MDA-MB-231 cells</td><td>ATCC</td><td>HTB-26</td><td>Breast cell line</td></tr><tr><td>Paraformaldehyde</td><td>Sigma, St.Louis, MO</td><td>158127-500G</td><td>cell fixative</td></tr><tr><td>Penicillin and streptomycin</td><td>Thermo Fisher, Waltham, MA</td><td>15140122</td><td>MCF10A / MDA-MB-231/HUVEC Media additive</td></tr><tr><td>Phosphate Buffered Saline 1X (PBS)</td><td>Thermo Fisher, Waltham, MA</td><td>7001106</td><td>Wash buffer for cells before trypsinization</td></tr><tr><td>Phosphate buffer saline 10X</td><td>Thermo Fisher, Waltham, MA</td><td>AM9625</td><td>immunofluorescence buffer component</td></tr><tr><td>Prolong gold antifade</td><td>Thermo Fisher, Waltham, MA</td><td>P36934</td><td>immunofluorescence mountant medium</td></tr><tr><td>Recombinant Human Epidermal Growth Factor, EGF</td><td>Peprotech, Rocky Hill, NJ</td><td>AF-100-15</td><td>MCF10A/ assay media component</td></tr><tr><td>Sodium Azide</td><td>Sigma, St.Louis, MO</td><td>S2002-25G</td><td>immunofluorescence buffer component</td></tr><tr><td>Sterile syringe (10 mL)</td><td>VWR, Radnor, PA</td><td>75846-757</td><td>bioprinting process</td></tr><tr><td>Tissue culture dish (10cm)</td><td>VWR, Radnor, PA</td><td>25382-166</td><td>monolayer cell culture</td></tr><tr><td>Triton X-100</td><td>Sigma, St.Louis, MO</td><td>T8787-250ML</td><td>immunofluorescence buffer component</td></tr><tr><td>Trypan blue 0.4%</td><td>Thermo Fisher, Waltham, MA</td><td>15250061</td><td>cell counter additive</td></tr><tr><td>Trypsin-EDTA 0.05%</td><td>Thermo Fisher, Waltham, MA</td><td>25300054</td><td>cell detachment</td></tr><tr><td>Tween -20</td><td>Thermo Fisher, Waltham, MA</td><td>85113</td><td>immunofluorescence buffer component</td></tr><tr><td>Vascular Endothelial Growth factor (VEGF&lt;sub&gt;165&lt;/sub&gt;)</td><td>Peprotech, Rocky Hill, NJ</td><td>100-20</td><td>HUVEC tube additive</td></tr><tr><td>Volocity 6.3 cell imaging software</td><td>PerkinElmer, Hopkinton, MA</td><td></td><td>Z stack compresser</td></tr></tbody></table>

direct bioprinting of 3d multicellular breast spheroids onto endothelial networks

バイオプリンティングは、生体組織アーキテクチャの重要な特徴をより良く再現する3Dヒトがんモデルを製造するための有望なツールとして浮上しています。現在のレイヤーごとの押出バイオプリンティングでは、個々の細胞が複雑な空間的および一時的な手がかりと共にバイオインクで押し出され、階層組織の自己集合を促進します。しかし、このバイオファブリケーション技術は、細胞間の複雑な相互作用、バイオインク、生化学的および生物物理学的手掛かりに依存しています。したがって、自己組立には数日または数週間かかる場合があり、特定のバイオインキを必要とすることがあり、複数の細胞タイプが関与している場合には必ずしも起こらない場合がある。そこで、様々なバイオインクで形成された3D乳房上皮スフェロイドを直接バイオプリントする技術を開発しました。バイオプリントされたプレフォーム3D乳房上皮スフェロイドは、印刷後の生存率と偏光アーキテクチャを維持した。さらに、3Dスフェロイドを血管内皮細胞ネットワークに印刷し、共培養モデルを作成しました。したがって、新しいバイオプリンティング技術は、従来のバイオプリンティング技術よりも低コストで柔軟性を持って、より生理学的に関連する3Dヒト乳房モデルを迅速に作成します。この多目的なバイオプリンティング技術は、追加のバイオインクで他の組織の3Dモデルを作成するために外挿することができます。

乳房上皮細胞は、マトリックス溶液上および2%マトリックス溶液を有する培養培地で5〜8日後に3Dスフェロイドに自己組織化すべきである。非腫瘍性MCF10A乳房上皮スフェロイドは円形に見え、中空中心を有し、インテグリンα6はスフェロイドの外縁に偏光している(図1、差し込みは中空の中心を示す)。高侵襲性MDA-MB-231乳癌上皮細胞は不規則なスフェロイドを形成する。回...

Watch this Scientific Journal Video about Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks at JoVE.com

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

このプロトコルの目的は、多細胞スフェロイドとして乳房上皮細胞を、薬物スクリーニング研究に使用できる3D乳房内皮共培養モデルを迅速に作成するために、多細胞性スフェロイドとして直接バイオプリントすることです。

内皮ネットワーク上の3D多細胞乳房スフェロイドの直接バイオプリンティング

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

direct-bioprinting-3d-multicellular-breast-spheroids-onto-endothelial

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

4.8K ビュー.  Drexel University. このプロトコルの目的は、多細胞スフェロイドとして乳房上皮細胞を、薬物スクリーニング研究に使用できる3D乳房内皮共培養モデルを迅速に作成するために、多細胞性スフェロイドとして直接バイオプリントすることです。

ビデオ: Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳がん研究のための潜在的機能 インビトロ モデルとしての乳腺上皮および内皮細胞スフェロイド

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

がん研究

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

三ディスペンサーデカルトプリンタを使用してBioprinted細胞構築物の生存率

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

生物工学

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

3D Bioprintingを用いたスフェロイドの機械的統合を示す心臓組織の創出

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

マイクロ流体工学バイオプリンティング血管組織や Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

マイクロ流体デバイスにおける組織モデルと perfusable の血管ネットワーク

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌細胞のスキャフォールドフリー3D培養におけるヘテロ多細胞間質細胞による浸潤の促進

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable アルギン酸/ゼラチン ゲル 3 Dの in Vitroモデル システム細胞スフェロイドの形成を誘導します。

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状構造の組織工学のためのコア/シェル印刷足場

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

3Dプリンティング技術を用いた血管フラップの作製

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

<meta charset="utf8"></head><body>プレート上の血管を使用した迅速な血管ネットワーク形成のための3Dバイオプリンティング

High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in coronary arteries and impaired function of the blood-brain barrier. Existing fabrication techniques do not adequately replicate the geometrical variation in vascular networks in the human body, which substantially influences disease progression; moreover, these techniques often involve multi-step fabrication procedures that hinder the high-throughput production necessary for pharmacological testing. This paper presents a bioprinting protocol for creating multiple vascular tissues with desired patterns and sizes directly on standard six-well plates, overcoming existing resolution and productivity challenges in bioprinting technology. A simplified fabrication approach was established to construct six hollow, perfusable channels within a hydrogel, which were subsequently lined with human umbilical vein endothelial cells to form a functional and mature endothelium. The computer-controlled nature of 3D bioprinting ensures high reproducibility and requires fewer manual fabrication steps than traditional methods. This highlights VOP's potential as an efficient high-throughput platform for modeling vascular permeability and advancing drug discovery.

<meta charset="utf8"></head><body>著者スポットライト:ハイスループット血管モデル作製のための自動バイオプリンティング

We present a protocol for high-throughput production of vascular channels with flexible sizes and desired patterns on a standard six-well plate using 3D bioprinting technology, referred to as vessels-on-a-plate (VOP). This platform has the potential to advance the development of therapeutics for the disorders associated with compromised endothelium.

サイズとパターンの柔軟性を備えた標準的な 6 ウェルプレートにおける血管透過性をモデル化するためのハイスループットバイオプリンティング法

Vascular permeability is a key factor in developing therapies for disorders associated with compromised endothelium, such as endothelial dysfunction in ...

内皮ネットワーク上の3D多細胞乳房スフェロイドの直接バイオプリンティング

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この動画の章

乳がん研究のための潜在的機能インビトロモデルとしての乳腺上皮および内皮細胞スフェロイド

三ディスペンサーデカルトプリンタを使用してBioprinted細胞構築物の生存率

3D Bioprintingを用いたスフェロイドの機械的統合を示す心臓組織の創出

マイクロ流体工学バイオプリンティング血管組織や Organoids

マイクロ流体デバイスにおける組織モデルと perfusable の血管ネットワーク

上皮癌細胞のスキャフォールドフリー3D培養におけるヘテロ多細胞間質細胞による浸潤の促進

Bioprintable アルギン酸/ゼラチンゲル 3 Dの in Vitroモデルシステム細胞スフェロイドの形成を誘導します。

管状構造の組織工学のためのコア/シェル印刷足場

3Dプリンティング技術を用いた血管フラップの作製

サイズとパターンの柔軟性を備えた標準的な 6 ウェルプレートにおける血管透過性をモデル化するためのハイスループットバイオプリンティング法

内皮ネットワーク上の3D多細胞乳房スフェロイドの直接バイオプリンティング

要約

さらに動画を探す

この動画の章

乳がん研究のための潜在的機能 インビトロ モデルとしての乳腺上皮および内皮細胞スフェロイド

三ディスペンサーデカルトプリンタを使用してBioprinted細胞構築物の生存率

3D Bioprintingを用いたスフェロイドの機械的統合を示す心臓組織の創出

マイクロ流体工学バイオプリンティング血管組織や Organoids

マイクロ流体デバイスにおける組織モデルと perfusable の血管ネットワーク

上皮癌細胞のスキャフォールドフリー3D培養におけるヘテロ多細胞間質細胞による浸潤の促進

Bioprintable アルギン酸/ゼラチン ゲル 3 Dの in Vitroモデル システム細胞スフェロイドの形成を誘導します。

管状構造の組織工学のためのコア/シェル印刷足場

3Dプリンティング技術を用いた血管フラップの作製

サイズとパターンの柔軟性を備えた標準的な 6 ウェルプレートにおける血管透過性をモデル化するためのハイスループットバイオプリンティング法

乳がん研究のための潜在的機能インビトロモデルとしての乳腺上皮および内皮細胞スフェロイド

Bioprintable アルギン酸/ゼラチンゲル 3 Dの in Vitroモデルシステム細胞スフェロイドの形成を誘導します。