저자는 &quot;유도된 pluripotent 줄기 세포의 행동을 제어하기위한 조직 공학 공사장 공중 발판&quot;NSERC 디스커버리 그랜트 402,462을 인정하고 싶습니다.

프로토콜을 시작하기 전에주의 : 섬유소는 혈액 유래 단백질이며, 따라서 적절한 안전 교육을 처리하기 전에 완료해야합니다. 이 프로토콜은 원하는 줄기 문화가 적절하게 그들이 시딩 준비하기 위해 시간이 딱 2 일 완료해야합니다. 얼마나 많은 섬유소가 트리스 세 35mm 배양 접시를 달다하는 계산의 측면에서 400가 포함된 1 24 잘 플레이트를 생산하기에 충분 될 것입니다 TBS 3 ML에 녹아있는 섬유소의 110-130 밀리그램을 포함 (TBS, 산도 7.4) 염분 버퍼 각 우물에 μl 섬유소의 공사장 공중 발판. 1. 일 하나 : 피브리노겐 솔루션 및 야간 투석 만들기 <ol><li> 냉장고에서 동결 건조된 섬유소를 꺼내서 그것은 실내 온도에 올 수 있도록 20 분 동안 앉아 보자. </li><li> 트리스의 추가 3 ML은 각 35mm 배양 접시에 염분 (TBS)을 버퍼. 12에서 20 농도에 이르기까지 피브리노겐 솔루션의 피브리노겐 수율의 각 접시는 2-3 MLMG / ML 및 필요한 섬유소의 총 금액은 이러한 approximations에 근거하여 계산하실 수 있습니다. </li><li> 약 100-130 MG의 피브리노겐 (FG)를 밖으로 무게와 각 접시에 TBS 선물의 표면에 뿌린다. 솔루션으로 이동하기 시작하는 피브리노겐 5 분 정도 기다립니다. 뚜껑과 배양 접시를 커버. </li><li> 37 품어 배양 접시는 ° C에서 2 시간 피브리노겐 완전히 TBS 용액에 들어갈 수 있습니다. </li><li> TBS와 젖은 투석 튜브 (7,000 MW가 잘려). 관 그리고 클램프 끝의 공간 하단 끝은 투석 클램프와 함께 종료합니다. </li><li> 투석 튜브로 요리에서 피브리노겐 솔루션을 피펫. 맨 끝 위에 접어 투석 클램프와 함께 종료 클램프. </li><li> 장소 투석 튜브는 TBS의 4리터 컨테이너에 피브리노겐 용액이 들어있는. 저속 (100 RPM)로 설정 볶음 플레이트를 사용하여 믹스 솔루션입니다. </li><li> 최소한 12 시간 동안 접시에 볶음에 하룻밤 솔루션을 Dialyze. TBS 솔루션은이 기간 동안 변경할 필요가 없습니다. </li></마셨다&gt; 2. 주 2 : 공사장 공중 발판으로 섬유소의 공사장 공중 발판 및 줄기 세포의 시딩의 중합 이 공사장 공중 발판이 줄기 세포 배양에 놓는되므로이 과정에 대해 설명하는 모든 단계가 무균 조직 배양 후드에서 수행해야합니다. <ol><li> 적절한 크기의 원뿔 튜브 (15 ML 50 ML 중)로 투석 튜브과 장소에서 피브리노겐 용액을 제거합니다. </li><li> 솔루션에 큰 불순물을 제거하는 5.0 μm의 주사기 필터로 필터 피브리노겐 솔루션입니다. 용액의 부피에 따라 여러 필터의 사용은 전체 솔루션을 처리 할 수​​ 있습니다. </li><li> 50mL 원뿔 튜브로 필터 0.22 μm의 가진 무균 필터 피브리노겐 솔루션입니다. 적절한 크기의 피펫을 사용하여 계산을 수행할 때 나중에 사용할 수있는 필터링 피브리노겐 용액의 볼륨을 결정합니다. 용액의 부피에 따라 여러 필터의 사용은 엔트를 처리하는 데 필요할 수 있습니다분노 솔루션입니다. </li><li> 분광 광도계를 사용하여 280 nm의에서 피브리노겐 용액의 흡광도를 측정합니다. 50 희석 요인은 1 아래 흡광도를 얻기 위해 종종 필요합니다. </li><li> 다음 방정식을 사용하여 피브리노겐 용액에 단백질 존재의 농도를 계산 : [MG / ML의 섬유소를] = (280 × 희석 계수) 1.55 인간 피브리노겐 13 280의 흡광 계수이다 ÷ 1.55. 다음 11.1 MG / 초기 볼륨을 기준으로 섬유소의 ML에 대한 솔루션의 농도를 희석하는 데 필요한 TBS의 양을 계산합니다. 섬유소 공사장 공중 발판에있는 단백질의 최종 농도는 중합시 10 밀리그램 / ML 될 것입니다. </li><li> 멸균 트롬빈과 염화칼슘 솔루션을 구합니다. 첫째, 15 μL 트롬빈 솔루션 추가 (농도 : 40 U / ML을 - 비계의 최종 농도는 2 U / ML 될 것입니다) 24 잘 접시의 첫 번째 행에있는 각 우물의 오른쪽. 다음, 50 MM 칼슘 chl 15 μL를 추가할각 우물의 왼쪽에 oride 솔루션입니다. 마지막으로 접시에 피브리노겐 솔루션의 270 μL를 추가합니다. 측면에서 솔루션의 혼합 수 있도록 전면에 다시 흔들어 뒤에 측면으로 접시를 흔들어. </li><li> 혼합물은 상온에서 5 분간 중합이 포함된 행을 보자. 그들이 중합으로 공사장 공중 발판은 더욱 불투명된다. </li><li> 섬유소의 공사장 공중 발판의 원하는 번호가 polymerized 때까지 단계 2.6 및 2.7을 반복합니다. </li><li> 마지막 5 분 인큐베이션 후, 공사장 공중 발판을 완전히 중합 수 있도록 37 ° C 배양기 안에서 접시를 놓습니다. </li><li> 한 시간 배양이 완료되면 다음 단계는 embryoid 시체 씨앗 발판을하는 것입니다. 20 μL pipettemen를 사용하여 섬유소 발판의 중심에서 개별 embryoid 기관과 장소를 선택합니다. 각 우물이 하나 embryoid 본문이 포함된 현미경으로 확인합니다. </li><li> 50 MM 염화칼슘 5 μL : 트롬빈 용액 5 μL (40 U / ML 농도) 추가각 잘으로 피브리노겐 용액의 90 μL 다음 각 embryoid 몸 위에 솔루션입니다. 솔루션 embryoid 시체를 캡슐 거품을 형성한다. </li><li> 중합을 보장하는 추가적인 시간 동안 다시 37 ° C 배양기에서 개최 플레이트. </li><li> 부화 후 37 ° C 배양기에 다시 각각 잘 장소 플레이트에 적절한 세포 배양 매체의 1 ML을 추가합니다. </li></ol> 3. 대표 결과 그림 1은 잘 embryoid 몸. 그림 2A와 함께 놓는 3D 섬유소 비계 문화 시스템을 포함하는 개인의 측면의 구조는 마우스 배아 fibroblast 피더 레이어에서 배양해되고 마우스 유도된 pluripotent 줄기 세포의 대표 이미지를 보여줍니다 보여줍니다. 이러한 세포들은 후 팔일 retinoic 산성 치료 프로토콜 (그림 2B) previousl 등을 사용하여 embryoid 기관, 신경 progenitors를 포함하는 세포의 집합체를 형성 유도하고그림 3은 3D 섬유소의 공사장 공중 발판의 내부 문화 3 일 후에 IPS - 파생 embryoid 신체의 모양을 보여주면서 y는 14 설명했다. 유사한 결과는 마우스 배아의 줄기 세포에게 7을 사용 이전에 취득했습니다. 또한이 방법이 문화 시스템의 다양성을 보여주는, retinoic 산성 및 purmorphamine 3D 섬유소의 공사장 공중 발판의 15 내부 관련된 다른 프로토콜을 사용하여 문화 IPS-파생 embryoid 신체에 생산 사용되었습니다. <img alt="1 그림." src="/files/ftp_upload/3641/3641fig1.jpg" /> 그림 1. 잘 embryoid 시신과 함께 3D 섬유소 비계 씨앗을 포함하는 개인의 측면을 보여주는 도식. <img alt="그림 2." src="/files/ftp_upload/3641/3641fig2.jpg" /> 그림 2. 마우스 유도 pluripotent 줄기 (IPS) 세포 배양 및 분화. neur로 이들 세포를 차별화하려면야외 phenotypes는 IPS 세포는 embryoid 기관 (EBS)라는 세포의 집계를 생성하기 위해 서스펜션에 배양해 있습니다. 이러한 EBS 나서 신경 분화를 유도하는 retinoic 산성 대우하고이 프로토콜은 이전에 배아 줄기 세포의 신경 분화를 유도하는 데 사용되었다. ) Undifferentiated 마우스 IPS 세포 식민지는 마우스 배아 fibroblast 피더 계층에서 배양해. B) 서스펜션 문화에 마우스 IPS 세포에서 파생된 Embryoid 시체는 신경 분화를 유도하는 retinoic 산성과 치료 후 일 8 일 촬영. 스케일 바는 100 μm의입니다. <img alt="그림 3." src="/files/ftp_upload/3641/3641fig3.jpg" /> 마우스 IPS의 그림 3. 예제 결과는 3D 섬유소 비계의 내부 문화 3 일 후에 시체를 embryoid. 세포는 섬유소 발판 내에 마이 그 레이션과 차별화하기 시작했다. 스케일 바 500 μm의입니다.

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저자가 공개하는 게 없다.

위에 설명된이 프로토콜은 마우스 배아 및 유도 pluripotent 줄기 세포에 대해, 특히 pluripotent 줄기 세포 배양을위한 3D 섬유소의 공사장 공중 발판을 생성하는 방법을 제공합니다. 이 3D biomaterial 기반의 문화 시스템은보다 정확하게 생체내에서 발견된 줄기 세포 틈새 시장을 모방한 것이었 그 결과로, 그것은 줄기 세포 분화 6 그들의 효과를 결정하기 위해 생물 학적 단서를 차단하는 ?...

장비 필요 : 분석 균형 산도 측정기 조직 문화 인큐베이터 (37 ° C에서 5 % CO 2) 플레이트 저어 분광 무균 조직 배양 후드 트리스는 (​​플러스 충분한 섬유소를 분해에 대해 4 패 필요) (산도 7.4) 염분을 버퍼 50 MM 염화칼슘 용액 멸균 원뿔 튜브 (15 또는 50 ML) 35mm 배양 접시 투석 튜브 (7,000 MW의 컷오프) 투석 클립 5.0 μm의 주사기 필터 개별 포장 멸균 0.22 μm의 주사기 필터 주사기 24 잘 조직 배양 플레이트 <table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 피브리노겐 (인간) </td><td> Calbioc가선을 두르다 </td><td> 341,578 </td><td></td></tr><tr><td> 트롬빈 (인간) </td><td> 시그마 </td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

줄기 세포가 자연스럽게 자주 줄기 세포 틈새 하나라고합니다 생체내에서 3D microenvironments를 발생에서 발견된다. 3D biomaterial 공사장 공중 발판의 내부 Culturing의 줄기 세포는 폴리스티렌뿐만 아니라 엔지니어링 교체 티슈 2에 대한 방법을 사용하여 전통적인 2D 문화 방법을 통해 이점을 제공, 정확하게 이러한 microenvironments을 모방하는 방법을 제공한다. 2D 조직 문화 polystrene은 세포 배양 실험의 대부분에 사용되고 있지만, 3D biomaterial의 공사장 공중 발판 더 밀접하게 환경에서 세포 극성의보다 정확한 창업을 활성화하고 부드러운 조직과 유사한 생화 학적 및 기계적 성질을 가진하여 생체내에서 발견 microenvironments를 복제할 수 있습니다. 3 다양한 자연적으로 파생과 합성 biomaterial의 공사장 공중 발판이 줄기 세포의 성장을 지원하기위한 3D 환경으로 조사되었습니다. 합성 공사장 공중 발판이 큰 R을 가지고 합성 수 있지만기계적 및 화학적 특성 앙과 자주보다 재현성을 가지고는 천연 biomaterials 종종 extracelluar 매트릭스와 세포 부착에 대한 바인딩 사이트를 포함하고 쉽게 세포 배양을 지원하는 결과로 발견된 단백질과 다당류로 구성되어있다. 혈장에서 얻은 단백질 섬유소를 polymerizing 제작한 섬유소 공사장 공중 발판은, 널리 시험 관내 및 생체내 4 두 조직 공학 응용 프로그램의 다양한 혐의로 조사되었습니다. 이러한 공사장 공중 발판은 치료적 요인에게 5를 전달하기위한 제어 릴리스 시스템을 통합하기 위해 다양한 방법을 사용하여 수정할 수 있습니다. 이전 작업은 공사장 공중 발판이 성공적으로 문화 배아 줄기 세포에 사용할 수있는이 발판 기반의 문화 체제가 6,7 내부에 놓는 줄기 세포의 분화에 관한 다양한 성장 요인의 효과를 차단하는 데 사용할 수있는 것으로 나타났습니다. 이 프로토콜 세부 polymerizin 과정g 섬유소는 트롬빈의 효소 활동을 사용하여 피브리노겐 솔루션에서 공사장 공중 발판. 과정은 중합을 억제 citrates를 제​​거하는 피브리노겐 솔루션을위한 야간 투석 단계를 포함하여 완료 2 일 소요됩니다. 이러한 세부적인 방법은 배아와 유도된 pluripotent 줄기 세포 배양을위한 최적의 것으로 확인된 피브리노겐 농도에 의존하고 있습니다. 다른 그룹은 더 이상 세포 유형과 어플 리케이션의 광범위한 섬유소의 공사장 공중 발판을 조사했습니다 -이 접근법 8-12의 다양성을 보여주는.

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

이 작품은 세부 3D 섬유소의 준비 culturing 및 plutipotent 줄기 세포를 구별을 위해 공사장 공중 발판. 이러한 공사장 공중 발판이 아니라 약물 전달 시스템을 포함하도록 수정으로 줄기 세포의 행동에 대한 다양한 생물 학적 화합물의 효과를 차단하는 데 사용할 수 있습니다.

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 ...

preparation-of-3d-fibrin-scaffolds-for-stem-cell-culture-applications

Research

JoVE Journal

Bioengineering

25.1K 조회수.  University of Victoria. 이 작품은 세부 3D 섬유소의 준비 culturing 및 plutipotent 줄기 세포를 구별을 위해 공사장 공중 발판. 이러한 공사장 공중 발판이 아니라 약물 전달 시스템을 포함하도록 수정으로 줄기 세포의 행동에 대한 다양한 생물 학적 화합물의 효과를 차단하는 데 사용할 수 있습니다.

비디오: Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

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 ...

연구

생체공학

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

3 차원 세포 배양 모델의 질량 분석 이미징을위한 준비 전략 샘플

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

관절 연골 세포 문화와 연골 생성을위한 3D 하이드로 겔 공사장 공중 발판

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

미세화 및 3D 미세 혈관의 조립

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

세포 배양 응용 프로그램에 대한 히드로 겔의 빛 매개 형성 및 패터닝

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

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

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 co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

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 ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3 차원 동적 문화 시스템과 다층된 중간 엽 줄기 세포 시트의 건설

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

정지 인간 조혈 줄기 세포를 유지하는 배양 방법

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

말초 신경 공학을 위한 그래핀 기반 3D 바이오하이브리드 하이드로겔 바이오잉크의 준비 및 특성화

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...