이 프로젝트의 재정 지원을위한 - 저자 켄트 주립 대학 (ReMedIKS 켄트 주에서 재생 의학 이니셔티브에 대한 공동 연구 보조금 및 지원) 감사의 말씀을 전합니다.

<ol>
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저자가 공개하는 게 없다.

액정 탄성체는 최근 인해 자극에 응답에 생체 세포 골격으로 연구되어왔다. 그들은 세포 골격으로 이상적인 플랫폼으로 입증되었다. 그러나 준비하고 새로운 LCE 발판을 설계 할 때 염두에 두어야 할 중요한 요소는 다공성이다. 침출 고체 (23) 또는 가스의 도입은 항상 균일 한 다공성 또는 완전히 상호 모공 발생하지 않습니다. 식각 할 수있는 금속 템플릿의 사용뿐만 아니라보다...

세포 연구 및 세포 부착 및 증식 1, 2, 3, 4, 5, 목표 조직 재생을위한 응용 프로그램을 위해 설계된 생물학적 생체 합성 재료의 몇 가지 예들이있다. 6,7 주문 이방성 분자와 외부 자극에 응답 할 수있는 액정 엘라스토머 (LCEs)라고도 생체 적합성 재료의 몇 가지 예,가 있었다. LCEs 광학 기능과 액정 분자 (8)의 순서, 9 엘라스토머의 기계적 성질과 탄성 결합 자극에 반응하는 물질이다. LCEs 외부 STIM에 응답하여 형상 변화, 기계적 변형, 탄성 거동 및 광학 특성을 이용할 수울리 (예., 열 스트레스, 빛 등) 10, 11, 12, 13, 14, 15, 16. 이전 연구는 액정 LCS (가) 세포 (4), (17)의 성장 방향을 감지 할 수 있음을 보여 주었다. LCEs 셀 목재 및 정렬을 포함한 생물학적으로 중요한 의학적 응용에 적합 할 수 있다고 가정하는 것이 가능하다. 우리는 이전에 &quot;스위스 치즈 유형&quot;다공성 형태 6, 18을 갖춘 스 멕틱 생체 적합성, 생분해 성, 주조 성형, 얇은 LCEs 필름의 준비를보고했다. 우리는 또한 세포 성장 (19) &lt;위한 발판으로 구상 형태와 네마 틱 생체 적합성 LCEs 준비SUP&gt; 20. 우리의 작업은 관심 (21)의 조직과 일치하는 재료의 기계적 특성을 조정하기위한되었다. 또한,이 연구는 탄성 중합체 세포의 상호 작용뿐만 아니라 탄성 중합체가 외부 자극을받습니다 세포 반응을 이해에 초점을 맞 춥니 다. 주요 문제는 탄성 중합체 매트릭스를 통해 세포 부착 및 침투를 허용하는 LCEs의 공극률을 조정하는 부분에 더 잘 질량 운송 하였다. 이러한 박막 (6)의 다공성 매트릭스의 대부분을 통해 세포 투과 허용하지만, 모든 세공이 완전히 상호 연결된 기공했다 또는 (균질)보다 일정한 크기를 가졌다. 우리는 구상 형태학와 생체 적합성 네마 틱 LCE 탄성 중합체 보도했다. 이러한 네마 엘라스토머 부착 세포의 증식을 허용하지만, 기공 크기는 10-30 ㎛의 방지, 또는 원거리 단에서 이들의 사용을 제한세포주 (19), (20)의 폭 넓은 다양한 엘라스토머. 쿵푸 등의 알에 의해 이전 작업. 은 &quot;희생&quot;금속 템플릿을 이용하여 그래 핀 발포체의 형성에 관한 얻어진 그라 발포체 선택된 금속 주형 (22)에 의해 지시 매우 일정한 다공성 형태를 한 것으로 나타났다. 이 방법론은 기공과 기공 크기의 전체 제어를 제공합니다. 다른 템플릿의 형성 발포 조제 전에 모양을 동시에, 금속 템플릿의 가단성 및 유연성을 허용한다. 이러한 재료 침출 23 가스 템플릿 (24) 또는 전기 방사 섬유 (25) 등의 다른 기술은, (26)는 다공질 재료의 제조 가능성을 제공하지만, 더 많은 시간이 어떤 경우에는 구멍 크기는 한정되어 걸리고이다 몇 마이크로 미터. 거품-like 3D LCEs 금속 템플릿 높은 셀 부하를 허용하여 제조; 개선 된 증식 속도; 공동 배양; 와, 적어도 마지막하지만, 더 나은 대중 교통 관리 (즉, 영양소, 가스, 폐기물)은 전체 조직 개발 (27)을 보장합니다. 폼 같은 3D LCEs 또한 셀 정렬을 개선하기 위해 표시; 이것은 세포 증식 및 세포 방향을 감지 LC 펜던트 관련된 대부분이다. LCE 내의 LC 잔기의 존재는 LCE 골격 내의 셀 위치에 대하여 셀의 정렬을 향상 보인다. 스트러트 함께 (접합부) (27)에 가입 여기서 명확한 방향은 관찰되지 동안 세포는 LCE의 스트러트 내에서 정렬. 전반적으로, 세포 지지체로서의 LCE 세포 골격 플랫폼 튜닝 엘라스토머 형태와 탄성 구체적 O 순서, 공간적인 배치를 만들 수 (개별) 세포 유형의 정렬을 지시하는이 기회를 제공한다생명체와 유사한 F 세포. 별개로 유지 장기 세포 성장 및 증식을 유도 할 수있는 발판을 제공하는 것을, 또한 셀 LCEs 방향 및 상호 작용을 실시간으로 수정 될 수 동적 실험을 허용한다.

<table><tbody><tr><td>1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane</td><td>Alfa Aesar</td><td>L16606</td><td>Silanizing agent</td></tr><tr><td>2-bis(4-hydroxy-cyclohexyl)propane</td><td>TCI</td><td>&lt;a href=&quot;http://www.tcichemicals.com/eshop/en/us/commodity/B0928/&quot;&gt;B0928&lt;/a&gt;</td><td>Reagent</td></tr><tr><td>2-chlorohexanone&amp;nbsp;</td><td>Alfa Aesar</td><td>A18613</td><td>Reagent</td></tr><tr><td>2-heptanone&amp;nbsp;</td><td>Sigma Aldrich</td><td>W254401</td><td>Solvent</td></tr><tr><td>2-propanol&amp;nbsp;</td><td>Sigma Aldrich</td><td>278475</td><td>Solvent</td></tr><tr><td>3-chloroperbenzoic acid, m-CPBA</td><td>Sigma Aldrich</td><td>273031</td><td>Reagent</td></tr><tr><td>4-dimethylaminopyridine</td><td>Alfa Aesar</td><td>A13016</td><td>Reagent</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole, DAPI&amp;nbsp;</td><td>Invitrogen</td><td>D1306</td><td>Nuclear Stain</td></tr><tr><td>5-hexynoic acid&amp;nbsp;</td><td>Alfa Aesar</td><td>B25132-06</td><td>Reagent</td></tr><tr><td>Acetic acid</td><td>VWR</td><td>36289</td><td>Solvent</td></tr><tr><td>Acetone</td><td>Sigma Aldrich</td><td>34850</td><td>Solvent</td></tr><tr><td>Alcohol 200 proof ACS Grade&amp;nbsp;</td><td>VWR</td><td>71001-866</td><td>Reagent</td></tr><tr><td>Benzene</td><td>Alfa Aesar</td><td>AA33290</td><td>Solvent</td></tr><tr><td>&amp;epsilon;-caprolactone&amp;nbsp;</td><td>Alfa Aesar</td><td>A10299-0E</td><td>Reagent</td></tr><tr><td>Chloroform</td><td>VWR</td><td>BDH1109</td><td>Solvent</td></tr><tr><td>Cholesterol</td><td>Sigma Aldrich</td><td>C8503</td><td>Reagent</td></tr><tr><td>Chromium(VI) oxide</td><td>Sigma Aldrich</td><td>232653</td><td>Reagent</td></tr><tr><td>Copper(I) iodide</td><td>Strem Chemicals</td><td>100211-060</td><td>Reagent</td></tr><tr><td>&lt;em&gt;D&lt;/em&gt;,&lt;em&gt;L&lt;/em&gt;-Lactide&amp;nbsp;</td><td>Alfa Aesar</td><td>L09026</td><td>Reagent</td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>320269</td><td>Solvent</td></tr><tr><td>Diethyl ether&amp;nbsp;</td><td>Emd Millipore</td><td>EX0190</td><td>Solvent</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide</td><td>Sigma Aldrich</td><td>270547</td><td>Solvent</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium, DEME&amp;nbsp;</td><td>CORNING Cellgo</td><td>10-013</td><td>Cell Media</td></tr><tr><td>Ethanol</td><td>Alfa Aesar</td><td>33361</td><td>Solvent</td></tr><tr><td>Formaldehyde&amp;nbsp;</td><td>SIGMA Life Science</td><td>F8775</td><td>Fixative</td></tr><tr><td>Fetal bovine serum, FBS&amp;nbsp;</td><td>HyClone</td><td>SH30071.01</td><td>Media Component</td></tr><tr><td>Filter paper, Grade 415, qualitative, crepe</td><td>VWR</td><td>28320</td><td>Filtration</td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G5516</td><td>Central node (3-arm)</td></tr><tr><td>Hexamethylene diisocyanate, HDI</td><td>Sigma Aldrich</td><td>52649</td><td>Crosslinker</td></tr><tr><td>Iron(III) chloride&amp;nbsp;</td><td>Alfa Aesar</td><td>12357</td><td>Etching agent</td></tr><tr><td>Isopropyl alcohol</td><td>VWR</td><td>BDH1133</td><td>Solvent</td></tr><tr><td>Methanol</td><td>Alfa Aesar</td><td>L13255</td><td>Solvent</td></tr><tr><td>&lt;em&gt;N,N&#039;&lt;/em&gt;-dicyclohexylcarbodiimide</td><td>Aldrich</td><td>D80002</td><td>Solvent</td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide</td><td>Sigma Aldrich</td><td>270547</td><td>Solvent</td></tr><tr><td>Nickel metal template</td><td>American Elements</td><td>Ni-860</td><td>Foam template</td></tr><tr><td>Neuroblastomas cells (SH-SY5Y)</td><td>ATCC</td><td>CRL-2266</td><td>Cell line</td></tr><tr><td>Penicillin streptomycin&amp;nbsp;</td><td>Thermo SCIENTIFIC</td><td>15140122</td><td>Antibiotics</td></tr><tr><td>Polyethylene glycol 2000, PEG</td><td>Alfa Aesar</td><td>B22181</td><td>Reagent</td></tr><tr><td>Sodium azide&amp;nbsp;</td><td>VWR</td><td>97064-646</td><td>Reagent</td></tr><tr><td>Sodium bicarbonate</td><td>AMRESCO</td><td>865</td><td>Drying salt</td></tr><tr><td>Sodium chloride</td><td>BDH</td><td>BDH9286</td><td>Drying salt</td></tr><tr><td>Sodium phosphate dibasic heptahydrate</td><td>Fisher Scientific</td><td>S-374</td><td>Drying salt</td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma Aldrich</td><td>S9638</td><td>Drying salt</td></tr><tr><td>Sodium sulfate</td><td>Sigma Aldrich</td><td>239313</td><td>Drying salt</td></tr><tr><td>Tetrahydrofuran</td><td>Alfa Aesar</td><td>41819</td><td>Solvent</td></tr><tr><td>Thiosulfate de sodium</td><td>AMRESCO</td><td>393</td><td>Drying salt</td></tr><tr><td>Tin(II) 2-ethylhexanoate</td><td>Aldrich</td><td>S3252</td><td>Reagent</td></tr><tr><td>Toluene</td><td>Alfa Aesar</td><td>22903</td><td>Solvent</td></tr><tr><td>Triethylamine</td><td>Sigma Aldrich</td><td>471283</td><td>Reagent</td></tr><tr><td>Trypsin</td><td>HyClone</td><td>SH30042.01</td><td>Cell Detachment</td></tr><tr><td>Olympus FV1000</td><td></td><td></td><td></td></tr></tbody></table>

synthesis of biocompatible liquid crystal elastomer foams as cell scaffolds for 3d spatial cell cultures

여기서는 3 차원, 생분해 성, 발포 셀 형상 지지체의 단계별 제조를 제시한다. 이러한 골격은 멕틱-A (SMA) 액정 엘라스토머 (LCEs) 결과, 측쇄 부속기로서 콜레스테롤을 갖춘 단위 성 블록 코 폴리머를 가교 결합하여 제조 하였다. 폼과 같은 골격은, 3 차원 세포 배양 지지체로 적합, 금속 템플릿을 이용하여 제조 상호 연결된 마이크로 채널이 있습니다. 금속 발포체의 종래 다공성 템플릿 필름이지만 질량 수송보다 잘 관리 (즉, 영양소, 가스 폐기물 비교뿐만 아니라 높은 세포 증식을 촉진하는 3 차원 세포 지지체의 엘라스토머 결과의 일반적인 구조의 결합 특성 등). 금속 템플릿의 특성상 거품 형상 (즉, 롤 또는 필름)의 간편한 조작을 가능하게하고 다른 세포 연구 다양한 기공 크기의 골격의 제조를위한 interconnec을 유지하면서템플릿의 테드 다공성 성격. 에칭 공정은 생체 적합성 및 생분해 성 특성을 보존, 엘라스토머의 화학적 성질에 영향을주지 않습니다. 우리는 세포의 성장과 증식을 촉진하면서 이러한 멕틱 LCEs는 광범위한 기간에 재배 할 때, 임상 적으로 복잡한 조직 구조의 연구를 수 있음을 보여준다.

이 보고서는 니켈 템플릿을 사용하는 세포 배양 용 지지체로서 다공성 3 차원 LCE의 제조 방법을 도시한다. 얻어진 3D LCE는 쉽게 세포의 침윤을 허용 복잡한 상호 채널 네트워크뿐만 아니라 더 적합 질량 수송 (27)를 보여줍니다. 그것은 세포가 완전히 상호 연결된 채널 네트워크에 침투 할 수 있고 또한 LCE 내에서 정렬 할 수있는 것을 알 수 있었다. 여기서,...

Watch this Scientific Journal Video about Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures at JoVE.com

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

본 연구는 3D, 생체 측쇄 액정 엘라스토머 (LCEs)에 기초한 생분해 성 발포 셀 형상 지지체를 제조하는 방법을 제공한다. 공 초점 현미경 실험은 거품 같은 LCEs가 세포 부착, 증식 및 C2C12s의 근육 모세포의 자연 정렬 할 수 있음을 보여준다.

3D 공간 세포 배양을위한 세포 발판으로 생체 적합성 액정 엘라스토머 거품의 합성

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

synthesis-biocompatible-liquid-crystal-elastomer-foams-as-cell

Research

JoVE Journal

Bioengineering

9.4K 조회수.  Kent State University.  본 연구는 3D, 생체 측쇄 액정 엘라스토머 (LCEs)에 기초한 생분해 성 발포 셀 형상 지지체를 제조하는 방법을 제공한다. 공 초점 현미경 실험은 거품 같은 LCEs가 세포 부착, 증식 및 C2C12s의 근육 모세포의 자연 정렬 할 수 있음을 보여준다. 

비디오: Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

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

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

3D 섬유소의 준비는 줄기 세포 배양 용도 공사장 공중 발판

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

연구

생체공학

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3 차원 세포 배양을위한 자기보고 발판

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

주 사용 Hydrogels 키토 산 기반 및 3D 세포 배양에서의 응용의 준비

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

화학

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin adhesives. Two different poly-(dimethyl siloxanes) (PDMS and soft skin adhesive (SSA)), have been used for in depth investigation of biological effects and adhesive characteristics. The composite films consist of a flexible backing layer and an adhesive top coating. Both layers have been manufactured by doctor blade application technique. In the present investigation, the adhesive behavior of the composite films has been investigated as a function of the layer thickness or a variation of the Young&#39;s modulus of the top layer. The Young&#39;s modulus of PDMS has been changed by varying the base to crosslinker mixing ratio. In addition, the thickness of SSA films has been varied from approx. 16 &#181;m to approx. 320 &#181;m. Scanning electron microscopy (SEM) and optical microscopy have been used for thickness measurements. The adhesive properties of elastomer films depend strongly on the film thickness, the Young&#39;s modulus of the polymers and surface characteristics. Therefore, normal adhesion of these films on glass substrates exhibiting smooth and rough surfaces has been investigated. Pull-off stress and work of separation are dependent on the mixing ratio of silicone elastomers.
Additionally, the thickness of the soft skin adhesive placed on top of a supportive backing layer has been varied in order to produce patches for skin applications. Cytotoxicity, proliferation and cellular adhesion of L929 murine fibroblasts on PDMS films (mixing ratio 10:1) and SSA films (mixing ratio 50:50) have been conducted. We have shown here, for the first time, the side by side comparison of thin composite films manufactured of both polymers and present the investigation of their biological- and adhesive properties.

A protocol for the manufacturing process of polymeric thin film composite structures possessing either different Young&#39;s moduli or thicknesses is presented. Films are produced for advanced cell culture studies or as skin adhesives.

세포 배양 및 피부 응용 프로그램에 대 한 영화 복합 실리콘 탄성 체를 얇은: 제조 및 특성

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

발생학

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

반전 콜로이드 결정체 폴리 (에틸렌 글리콜) 비계의 제작 : 간 조직 공학을위한 삼차원 세포 배양 플랫폼

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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.

3 차원 세포 배양을위한 폐 세포 외 매트릭스에서 조정 히드로 겔

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

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

조직 특이 적 세포 배양 및 전달 플랫폼과 같은 세포 외 기질 유래 거품과 마이크로 담체의 제조

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

3 차원 펩 티 드 건설 기계에 포유류 세포 배양

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

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.

캡슐화 및 3d에서 셀의 Immunostaining에 대 한 엘라 스 틴 같은 단백질 Hydrogels의 생산

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

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

마이크로젤 로드의 상호 연결된 거대 다공성 3D 스캐폴드

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...