وقد تم تمويل هذا المشروع من قبل المعاهد الوطنية للصحة (R21EB013717) وIUPUI OVCR (RSFG). المؤلف يشكر السيدة هان شي لمساعدتها التقنية.

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الإعلان عن أي تضارب في المصالح.

البروتوكول وصف تفاصيل يعرض على تغليف سهلة من الخلايا في ثيول-إيني الهلاميات المائية التي شكلتها خطوة نمو بلمرة ضوئية المنشأ. في حين تم استخدام نسبة 1:1 من متكافئة من نوربورنين إلى المجموعات الوظيفية ثيول في هذا البروتوكول، يمكن تعديل نسبة اعتمادا على التجارب. بالإضاف...

الهلاميات المائية هي البوليمرات ماء crosslinked مع إمكانات استثنائية كمواد سقالات للإصلاح وتجديد أنسجة 1-3 من محتوى الماء عالية من الهلاميات المائية يسمح نشر سهلة من الأوكسجين والمواد المغذية وتبادل المنتجات الأيضية الخلوية، والتي تعتبر حاسمة للحفاظ على بقاء الخلية. وبالإضافة إلى ذلك، الهلاميات المائية يحملون ممتازة لإطلاق وتسليم خلية تسيطر عليها بسبب tunability العالية .. 2 الهلاميات المائية الاصطناعية مثل تلك المحضرة من بولي (إيثيلين جليكول) (PEG) تستخدم بشكل متزايد في تطبيقات هندسة الأنسجة، إلى حد كبير بسبب cytocompatibility بهم، الأنسجة مثل مرونة، وtunability عالية في المواد الخصائص الفيزيائية والميكانيكية 4-6 على الرغم من أن منصة هيدروجيل شيوعا، وقد أظهرت الدراسات أن PEG diacrylate (PEGDA) الهلاميات المائية التي شكلتها سلسلة النمو photopolymerizations لديهم ميل إلى تلف خلايا مغلفة الدورينانوغرام يشابك شبكة والتغليف في الخلية الموقع .. 7 ويعزى إلى حد كبير إلى تلف الخلايا الراديكالية الأنواع التي تم إنشاؤها بواسطة جزيئات photoinitiator، الذي نشر من خلال مجموعات الفينيل على PEGDA لسلاسل البوليمر تشعبي في الهلاميات المائية. للأسف، هذه الأنواع المتطرفة أيضا أن يسبب تلف الخلايا الضغوط والتغليف خلال الخلية، وخاصة بالنسبة للراديكالية الخلايا الحساسة للالبنكرياس β مثل خلايا 8-10 من أجل الحصول على أعلى حجم شبكة لأفضل نشرها وبقاء الخلية، والأوزان الجزيئية أعلى PEGDA وغالبا ما تستخدم لتغليف الخلايا. هذا، ومع ذلك، يعرض للخطر حركية البلمرة ويسبب خصائص هلام دون المستوى الأمثل البيوفيزيائية. 7،11،12 بالإضافة إلى عيوب المذكورة أعلاه، فإنه من الصعب للغاية لاستعادة هياكل الخلية من الهلاميات المائية PEGDA نظرا لطبيعة التغاير وغير القابلة للتحلل، من شبكات crosslinked. في حين يمكن إدراج البروتيني حساسة الببتيداتفي العمود الفقري macromer PEG لتقديم خامل غير ذلك الهلاميات المائية PEGDA حساسة للانشقاق الأنزيمية، وغالبا ما يستخدم اقتران الكواشف باهظة الثمن وشبكات الناتجة لا تزال تحتوي على درجة عالية من عدم التجانس نظرا لطبيعة سلسلة البلمرة النمو. 13-15 في الآونة الأخيرة، وقد ثبت PEG-الببتيد الهلاميات المائية شكلت عبر بلمرة ضوئية المنشأ ثيول-إيني خطوة نمو لعرض خصائص تفضيلية لتغليف الخلايا على الهلاميات المائية التي شكلتها سلسلة النمو بلمرة ضوئية المنشأ. 7 وحركية الهلام متفوقة من ثيول-إيني الهلاميات المائية ويعزى إلى فوق &quot; &quot;طبيعة التفاعل بين ثيول وظائف إيني. مقارنة سلسلة البلمرة من النمو PEGDA، ثيول-إيني رد فعل أقل الأكسجين مما يؤدي إلى تثبيط معدل أسرع الهلام. 16،17 ثيول-إيني الهلاميات المائية أيضا أعلى كفاءة البلمرة وخصائص أفضل هلام البيوفيزيائية مقارنة الهلاميات المائية PEGDA سلسلة النمو، 7 ، 18 &lt;/ سوب&gt; مما يؤدي إلى تلف الخلايا التي تسببها الأنواع محدودة جذرية خلال بلمرة ضوئية المنشأ. سابقا، ثيول-إيني الهلاميات المائية التي شكلتها الذراع 4-macromer (PEG4NB) PEG-نوربورنين ومكرر السيستين الببتيد تحتوي على crosslinkers، مثل البروتياز حساسة استخدمت لتغليف الخلايا الببتيدات. 7،18 tunability العليا شبكات هيدروجيل يقدم PEG 3D المكروية مرنة ويمكن السيطرة عليها عن التحقيق في بقاء الخلية والنشاط، في حين أن استخدام البروتيني تراعي تسلسل الببتيد يوفر طريقة خفيفة لاسترداد بنيات الخلية تشكلت بشكل طبيعي داخل الهلاميات المائية. في هذا البروتوكول خطوة نستخدم النمو photopolymerized ثيول-إيني الهلاميات المائية ملفقة باستخدام الذراع 4-PEG-نوربورنين (PEG4NB) وcrosslinker الببتيد كيموتربسين حساسة (CGGY ↓ C) لتغليف الخلايا β MIN6. هذا البروتوكول بالتفصيل منهجية لدراسة تقنيات تشكيل البقاء، وانتشار كروي من MIN6β-cells في ثيول-إيني الهلاميات المائية. نحن نقدم طريقة لزيادة β خلايا الانتعاش كروي وتوصيف الأجسام الشبه الكروية البيولوجي للشفاء.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> 4-PEG الذراع (20kDa) </td><td> Jenkem التكنولوجيا USA </td><td> 4ARM-PEG-20K </td><td></td></tr><tr><td> Fmoc الأحماض الأمينية </td><td> Anaspec </td><td></td><td></td></tr><tr><td> لايف / الميت عدة خلايا الجدوى </td><td> إينفيتروجن </td><td> L3224 </td><td> يتضمن Calcein AM إيثيديوم وhomodimer-1 </td></tr><tr><td> كاشف AlamarBlue </td><td> عبد Serotec </td><td> BUF012 </td><td></td></tr><tr><td> CellTiter بالشبكة كاشف </td><td> Promega </td><td> G7570 </td><td></td></tr><tr><td> DPBS </td><td> Lonza </td><td> 17-512F </td><td> و +2 دون الكالسيوم المغنيسيوم +2 </td></tr><tr><td> HBSS </td><td> Lonza </td><td> 10547F </td><td> بدونCA +2 +2 والماغنسيوم </td></tr><tr><td> ارتفاع الجلوكوز DMEM </td><td> Hyclone </td><td> SH30243.01 </td><td></td></tr><tr><td> FBS </td><td> Gibco </td><td> 16000-044 </td><td></td></tr><tr><td> درجة التأثير للمضادات الحيوية </td><td> إينفيتروجن </td><td> 15240-062 </td><td></td></tr><tr><td> β-المركابتويثانول </td><td> سيغما الدريخ </td><td> M7522-100ML </td><td></td></tr><tr><td> التربسين EDTA- </td><td> إينفيتروجن </td><td> 15400-054 </td><td></td></tr><tr><td> التربسين خالية α-كيموتربسين </td><td> ورثينجتون شركة بيوكيميائية </td><td> LS001432 </td><td></td></tr><tr><td> الماوس Inusin ELISA عدة </td><td> Mercodia </td><td> 10-1247-01 </td><td></td></tr><tr><td> 1 مل الحقنة </td><td> BD العلوم البيولوجية </td><td></td><td></td></tr></tbody></table>

generation and recovery of &beta;-cell spheroids from step-growth peg-peptide hydrogels

الهلاميات المائية هي البوليمرات ماء crosslinked التي توفر المكروية ثلاثية الأبعاد مع الأنسجة مثل مرونة ونفاذية عالية للخلايا ذات الصلة زراعة الأنسجة أو علاجية. يتزايد استخدام الهلاميات المائية المحضرة من بولي (إيثيلين جليكول) (PEG) المشتقات لمجموعة متنوعة من التطبيقات هندسة الأنسجة، ويرجع ذلك جزئيا إلى ممتلكاتهم والانضباطي cytocompatible. في هذا البروتوكول، ونحن تستخدم ثيول-إيني خطوة نمو photopolymerizations الى افتعال PEG-الببتيد الهلاميات المائية للتغليف البنكرياس MIN6 الخلايا البائية. وتم تشكيل المواد الهلامية التي كتبها 4-الذراع macromer (PEG4NB) PEG-نوربورنين وcrosslinker الببتيد كيموتربسين حساسة (CGGYC). طبيعة ماء وغير تلوث من PEG يقدم المكروية cytocompatible لبقاء الخلية والانتشار في 3D، في حين أن استخدام تسلسل الببتيد كيموتربسين حساسة (C GGY ↓ C، سهم يشير إلى الانقسام انزيم موقع، في حين كيسة محطةأضيفت مخلفات eine للثيول-إيني يشابك) يسمح الانتعاش السريع للبنيات الخلية تشكيل هيدروجيل داخل. بروتوكول التالية بالتفصيل تقنيات ل: (1) تغليف من MIN6 β-cells في ثيول-إيني الهلاميات المائية، (2) نوعية وكمية المقايسات بقاء الخلية لتحديد بقاء الخلية والانتشار، (3) استرداد الأجسام الشبه الكروية الخلية باستخدام كيموتربسين بوساطة هلام تآكل، و(4) التحليل البنيوي والوظيفي للالأجسام الشبه الكروية استردادها.

<html> الأرقام 1-4 نتائج عرض ممثل من أجل البقاء، التغليف، والانتشار، وتشكيل كروي، والانتعاش كروي في ثيول-إيني الهلاميات المائية. الشكل 1 يبين التخطيطي رد فعل (1) بلمرة ضوئية المنشأ ثيول-إيني خطوة النمو باستخدام PEG4NB وCGGYC، و( 2) تآكل كيموتربسين هلام بوساطة ا?...

Watch this Scientific Journal Video about Generation and Recovery of Î²-cell Spheroids From Step-growth PEG-peptide Hydrogels at JoVE.com

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

بروتوكول التالية توفر تقنيات التغليف البنكرياس β-cells في الهلاميات المائية PEG-الببتيد خطوة نمو ردود الفعل التي شكلتها الصورة مزدوجا فوق ثيول-إيني. هذا البرنامج ليس فقط المواد يقدم المكروية لتغليف الخلايا cytocompatible، ولكن أيضا يسمح للمستخدم التحكم الانتعاش السريع للهياكل الخلية تشكلت في الهلاميات المائية.

جيل واسترداد خلايا β الأجسام الشبه الكروية من الهلاميات المائية PEG-الببتيد الخطوة النمو

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

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

14.1K Views.  Indiana University - Purdue University at Indianapolis. بروتوكول التالية توفر تقنيات التغليف البنكرياس β-cells في الهلاميات المائية PEG-الببتيد خطوة نمو ردود الفعل التي شكلتها الصورة مزدوجا فوق ثيول-إيني. هذا البرنامج ليس فقط المواد يقدم المكروية لتغليف الخلايا cytocompatible، ولكن أيضا يسمح للمستخدم التحكم الانتعاش السريع ل...

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

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

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

إسقاط خلية بسيطة الشنق بروتوكول الثقافة للجيل 3D الأجسام الشبه الكروية

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

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

ألجينات الهلاميات المائية للثقافة الجهاز ثلاثي الأبعاد من المبيضين وناقلة البيضات

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

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.

الانضباطي الهلاميات المائية من الرئوي خارج الخلية مصفوفة للثقافة خلية 3D

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

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.

إنتاج بروتين الايلاستين مثل الهلاميات المائية للتغليف وإيمونوستينينج من الخلايا في 3D

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.

سلالة تسيطر عليها من هيدروجيلس 3D تحت التصوير المجهري لايف

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

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

التصنيع الحيوي بمساعدة الأكسجين بمساعدة الأكسجين بأسعار معقولة للكرويات متعددة الخلايا

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

Fragmenting Bulk Hydrogels and Processing into Granular Hydrogels for Biomedical Applications

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have many advantageous properties, including injectability, microscale porosity, and tunability by mixing multiple microgel populations. Methods to fabricate microgels often rely on water-in-oil emulsions (e.g., microfluidics, batch emulsions, electrospraying) or photolithography, which may present high demands in terms of resources and costs, and may not be compatible with many hydrogels. This work details simple yet highly effective methods to fabricate microgels using extrusion fragmentation and to process them into granular hydrogels useful for biomedical applications (e.g., 3D printing inks). First, bulk hydrogels (using photocrosslinkable hyaluronic acid (HA) as an example) are extruded through a series of needles with sequentially smaller diameters to form fragmented microgels. This microgel fabrication technique is rapid, low-cost, and highly scalable. Methods to jam microgels into granular hydrogels by centrifugation and vacuum-driven filtration are described, with optional post-crosslinking for hydrogel stabilization. Lastly, granular hydrogels fabricated from fragmented microgels are demonstrated as extrusion printing inks. While the examples described herein use photocrosslinkable HA for 3D printing, the methods are easily adaptable for a wide variety of hydrogel types and biomedical applications.

This work describes straightforward, adaptable, and low-cost methods to fabricate microgels with extrusion fragmentation, process the microgels into injectable granular hydrogels, and apply the granular hydrogels as extrusion printing inks for biomedical applications.

تجزئة المواد الهلامية السائبة ومعالجتها إلى هيدروجيل حبيبي للتطبيقات الطبية الحيوية

Granular hydrogels are jammed assemblies of hydrogel microparticles (i.e., &#34;microgels&#34;). In the field of biomaterials, granular hydrogels have ...

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

كرويات الكبد البشري من الدم المحيطي لدراسات أمراض الكبد

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

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.

توليد ، فحص عالي الإنتاجية ، وبنوك حيوية للكرويات القلبية المشتقة من الخلايا الجذعية متعددة القدرات التي يسببها الإنسان

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

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

تخليق الهلاميات المائية مصفوفة الغضروف خارج الخلية

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...