我们要感谢史密斯菲尔德农场捐赠了完整的猪肺组织。我们也想感谢胡扬博士，唐克里斯蒂娜博士和VCU整形外科部允许我们使用他们的设备。水凝胶和组织样品进行SEM准备在解剖学和神经生物学显微镜基金的VCU部支持的，一部分是由NIH-NINDS中心核心格兰特5 P30 NS047463的资金，部分通过资金形式NIH-NCI癌症中心支援津贴P30 CA016059。在VCU纳米技术核心表征基金（NCC）的SEM图像。这项工作是由美国国家科学基金会，CMMI 1351162资助。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 解 </td><td> 无菌过滤器 </td><td> 说明 </td></tr><tr><td> DIH 2 O </td><td>是</td><td> DIH 2 O;无菌过滤</td></tr><tr><td> 0.1％的Triton X-100溶液</td><td>是</td><td>在通风柜100微升的Triton-X 100溶液加至100毫升DIH 2 O和搅拌至溶解; 无菌过滤器。 </td></tr><tr><td> 2％的脱氧胆解决方案</td><td>是</td><td>在通风橱中添加2克脱氧胆酸钠溶液 每100毫升DIH 2 O和搅拌至溶解;无菌过滤器</td></tr><tr><td> 1M NaCl的</td><td>是</td><td> 58.44克氯化钠加入1升DIH 2 O的;搅拌至溶解; 无菌过滤器</td></tr><tr><td> DNA酶的解决方案</td><td>是</td><td>添加12000单位DNase的1升DIH 2 O;添加0.156克硫酸镁 （无水），和0.222克CaCl 2;搅拌至溶解;无菌过滤器</td></tr><tr><td> PBS </td><td>是</td><td>结合27.2克的 Na 2 HPO 4·7H 2 O（二钠七水合物） 80克氯化钠和2g氯化钾用10L DIH 2 O;搅拌至溶解;调节pH值至7.4;无菌过滤器</td></tr></tbody></table> 表1: 所需的组织脱细胞的解决方案。使上述对脱细胞过程的解决方案。保存在4℃。约将需要一个猪肺2.5升。 1.水凝胶形成<ol><li> 猪肺脱细胞（改编自引用9,10） <ol><li>苦练内功， 整块正常猪肺，从屠宰场，与心脏和VASCulature完好无损。 </li><li>用组织剪或手术刀解剖远离心脏，血管切割尽可能靠近心脏，血管其余将用于灌注后面的步骤中使用。 </li><li>小心解剖距离围绕气管，支气管，和使用解剖刀或剪刀脉管结缔组织。 注:确定最佳肺通过选择肺不通过胸膜大切口或穿刺和大量肺不张或血管闭塞可能妨碍脱细胞过程的有效性以用于程序的其余部分。 </li><li>切去次优肺留下附着到气管尽可能多支气管（心脏，结缔组织和除去肺的凸起可设置的）。应该有一个肺保持附着到气管和血管。 注意:如果需要保留的部分组织学。 9 </li><li>关闭用夹子固定或缝合至P断开支气管r事件多余的回流。 </li></ol><ol start="6"><li>直到需要（ 表1）在4℃准备脱细胞的解决方案和冷藏。 </li><li>使用插管以适合肺动脉，通过两个肺动脉和气管灌注肺组织用DI水3次手泵。第一血管灌注每次。与周围的1L到脉管和1.5升进气管每个灌注开始，但是作为细胞碎片除去更多的液体可通过该系统来灌注。 </li><li>灌注两个脉管和气管用Triton X-100溶液。 </li><li>淹没肺组织中的Triton X-100解决方案，在4℃下24小时。 </li><li>灌注血管和气管离子水3次冲洗。 </li><li>既灌注血管和气管与脱氧胆酸的解决方案。 </li><li>浸没在脱氧胆酸盐的组织切片，在4℃下24小时。 </li><li>灌注血管和气管离子水3次冲洗。 </li><li>灌注两个脉管和气管用NaCl溶液。 </li><li>淹没在过滤的NaCl溶液用于组织在4℃1小时。 </li><li>灌注血管和气管离子水3次冲洗。 </li><li>既灌注血管和气管用DNase解决方案。 </li><li>淹没在过滤的DNA酶溶液中的组织在4℃1小时。 </li><li>灌注两个脉管和气管5次，用PBS。 </li><li>解剖走明显的软骨组织，气管和传导气道，只留下呼吸的区域（主要是周边地区）的所有直径管2mm或更大（主要围绕肺门和肺的中间部分找到）。 </li><li>解剖组织成1英寸的部分或更小。的组织的取向并不重要此步骤。 </li><li>在50ml锥形管中倒出多余的液体和地方组织。冷冻在-80℃下的组织。 注:保留部分组织学，以确保去除细胞和细胞debri的S，如果需要的话。我们使用H＆E，α半乳糖苷酶的PicoGreen，羟脯氨酸，茚三酮，阿新蓝，SDS-PAGE和质谱表征。 9 </li></ol></li><li> 隆处理 <ol><li>拆除含脱细胞肺组织冷冻管盖。 </li><li>将滤纸在管口并用橡皮筋固定。 注:管材与内容仍然应该被冻结，否则置于-80℃直至冻结。 </li><li>冻干组织，直到所有多余的液体是根据制造商的方向走了，用冷冻干燥机。储存在-80°C，直到准备磨。 </li><li>才开始加液氮冷冻研磨机冷却绝缘和内部元件的工作条件。 </li><li>从磨筒中取出磁磨棒并加入组织覆盖底部。 </li><li>更换磨棒，并添加松散的组织。该厂酒吧还是应该自由移动。 </li><li>关闭工厂管和PLACE在冷冻研磨机。 </li><li>填写液氮最大填充线。 </li><li>冷冻粉碎机所有组织成细粉（约5分钟，〜600分钟嵌塞速率-1）时 ，在用不锈钢冲击的聚碳酸酯圆筒以及根据制造商的说明使用冷冻粉碎机不锈钢端塞。储存在-80°C，直到准备使用。 </li></ol></li><li> 微多孔凝胶形成（8毫克/毫升）（改编自引用9,11） <ol><li>添加1％（重量/体积）的脱细胞的粉末和0.1％的（重量/体积）胃蛋白酶的0.01M HCl中，在恒定搅拌下（应能看到流在液体的顶层），在室温下，为48小时。 注:粉末带有静电，因此添加盐酸的粉末，得到机会洗过量离管壁之后。 </li><li>消化48小时后，将溶液和试剂在冰上5分钟。 </li><li>使用冷冻的10％（体积/体积）0.1M NaOH中，一个ð11.11％（体积/体积）的10倍的PBS（以使整个溶液为1x浓度），使消化的蛋白溶液，以7.4生理pH。 注意:溶液现在可以储存在4℃下长达一周。执行如果需要使用流变9凝胶动力学。 </li></ol></li></ol> 2.交联水凝胶，以提高机械强度<ol><li>通过京尼平粉末的所需量溶解在10％DMSO中制备的1％，0.1％和0.01％w / v的京尼平交联溶液的解决方案。 </li><li>涡的解决方案，每15分钟1小时。 </li><li>加京尼平溶液以覆盖先前已经组装在部分1.3.4 ECM凝胶（100μL用于96孔板的每个孔）。 </li><li>离开每个ECM凝胶在37℃下交联24小时。 </li><li>冲洗的ECM凝胶用PBS 3次，直到洗涤液不再蓝色。然后将交联的水凝胶将准备用于进一步表征和CELL文化研究。 </li></ol> 3.细胞培养与微孔凝胶<ol><li> 二维涂层 <ol><li>从1.3.3使用溶液中，添加20微升的预凝胶溶液到96孔非治疗的组织培养板的每个孔中。 </li><li>在4℃下冷藏过夜，使蛋白质吸附于板。 </li><li>吸预凝胶溶液，用PBS冲洗。 </li><li>传代细胞9。 </li><li>加10,000个细胞/ cm 2至孔中。 </li><li>增加媒体每孔100μl，并在37℃下孵育。 </li></ol></li><li> 三维细胞培养9 <ol><li>从1.3.3采用溶液，悬浮沉淀的细胞，以100万个细胞/ ml的预凝胶溶液中的浓度。 </li><li>迅速分配16微升预凝胶细胞悬液到96孔平板的每个孔中，以形成一个5〜50微米厚的水凝胶为三维细胞培养物。 </li><li>孵育在37℃，30分的凝胶形成。 </li><li>加入100微升的媒体到形成水凝胶的顶部，并在37℃孵育。 </li></ol></li><li> 在变刚度凝胶的三维细胞培养 <ol><li>从1.3.3使用溶液，吸管100微升的预凝胶溶液加入到96孔板的每个孔中。 </li><li>孵育在37℃下30分钟进行凝胶形成。 注意:交联从第2部分的步骤可在此使用。 </li><li>传代细胞9。 </li><li>加10,000个细胞/ cm 2至孔中。 </li><li>增加媒体每孔100μl，并在37℃下孵育。 </li></ol></li></ol>

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作者什么都没有透露。

之一的生物学的积分方面是自组织分子成执行特定任务层次结构。 13在实验室中，自组装取决于许多因素，如盐浓度，pH和消化时间。如图所示，自组织的水凝胶形式时溶解蛋白质返回到生理温度。形成的水凝胶是能够促进在体外的细胞附着和增殖的。 从ECM生物物理信号的细胞应答不能使用的聚合物进行研究。生物物理信号组织特异性细胞应答不能使用其他组?...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table><tbody><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher Scientific</td><td>BP151-100</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>D6750-100g</td><td>Use in hood with eye protection and gloves.</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506-500g</td><td>None</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C1016-500g</td><td>None</td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td>None</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>258148-500ML</td><td>Use with eye protection and gloves.</td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich</td><td>P6887-5G</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>BP359-500</td><td>Use with eye protection and gloves.</td></tr><tr><td>Genipin</td><td>Wako Chemicals</td><td>078-03021</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>PBS 10x</td><td>Quality Biological</td><td>119-069-151</td><td>None</td></tr><tr><td>PBS</td><td>VWR</td><td>45000-448</td><td>None</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>8519</td><td>N/A</td></tr><tr><td>Hand pump</td><td>Fisher Scientific</td><td>10-239-1</td><td>N/A</td></tr><tr><td>Graduate Beaker</td><td>VitLab</td><td>445941</td><td>N/A</td></tr><tr><td>Cryomill</td><td>SPEX</td><td>6700</td><td>Use cryogloves and eye protection.</td></tr><tr><td>Lyophilizer</td><td>FTS FlexiDry</td><td></td><td>Use gloves.</td></tr><tr><td>Rheometer</td><td>Discovery</td><td>HR-2</td><td>Use gloves and eye protection.</td></tr></tbody></table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

在这里，我们提出了体外肺细胞培养建立的多组分细胞培养水凝胶的方法。用健康途中从猪，大鼠，或小鼠集团肺组织开始，组织灌注并在随后的化学洗涤剂浸没以除去细胞碎片。处理之前和之后的组织的组织学比较证实除去双链DNA和α-半乳糖苷酶染色的95％以上表明大部分细胞碎片除去。脱细胞后，组织是冻干然后cryomilled成粉末。基质粉末在酸性胃蛋白酶消化溶液消化48小时，然后中和形成的预凝胶溶液中。该预凝胶溶液的凝胶化可通过温育在37℃下被诱导，并且可以立即使用以下中和或储存在4℃下长达两周。涂层可以用在未处理板c中的预凝胶溶液来形成埃尔附件。细胞可以悬浮在之前自组装，实现了3D培养的预凝胶，电镀形成的凝胶从该细胞可以通过在支架迁移，或镀在涂层的表面上。改变的策略提出可影响凝胶化温度，强度，或蛋白质片段的大小。以外的水凝胶的形成，所述水凝胶的刚度可使用京尼平可以增加。

使用这种方法，我们已经产生从正常猪，大鼠和小鼠的肺（ 图1）的水凝胶。处理肺提供一种估计分别5毫克，40毫克，和10g的ECM粉末。该过程的概要示于图2。在此过程中关键的可视化包括:清洗后脱氧胆肺的白色外观;该预凝胶形成后，溶液应是不透明的，如果保存在4℃的溶液应几个月出现均匀。我们已经包括了基本的故障排除表，以帮助确定在整...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

这是创建从肺的细胞外基质的三维细胞培养物的支架的方法。完好肺被加工成可支持细胞以三维生长的水凝胶。

可调水凝胶由肺细胞外基质的三维细胞培养

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

tunable-hydrogels-from-pulmonary-extracellular-matrix-for-3d-cell

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.6K 次观看.  Virginia Commonwealth University. 这是创建从肺的细胞外基质的三维细胞培养物的支架的方法。完好肺被加工成可支持细胞以三维生长的水凝胶。 

视频: Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

科研

生物工程

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

生物化学

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

生物印刷细胞化构造使用组织特异性水凝胶生物油墨

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

水凝胶阵列可提高 3D 肿瘤模型中基质成分和治疗药物筛选效果的通量

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

光调谐生物打印水凝胶，用于探测细胞对 ECM 硬化的反应

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

作者聚焦：使用 3D 打印光可调谐水凝胶了解慢性肺病 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

3D生物打印光可调水凝胶研究成纤维细胞活化

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...