저자 감사 토니 파머와 H. 간디 (스탠포드 신경외과) 그림 4에서 murine NPCs 벡터 아트를 제공 하는 사용 하 고 크리에이 티브 커먼즈 저작자 표시 3.0 Unported 라이센스 (https://creativecommons.org/ 아래 Servier 의료 예술에서 적용 licenses/by/3.0/legalcode)입니다. 이 작품의 일부에는 스탠포드 나노 공유 시설 (SNSF), 수상 ECCS-1542152에서 국립 과학 재단에 의해 지원 되는 수행 되었다. N.A.S. 지원 국립 과학 연구소에서의 일반 의료 건강의 국가 학회 (32 GM 008412)의 인정합니다. C.M.M. 사전 박사 친목 (F31 EB020502) 그리고 Siebel 학자 프로그램 NIH NRSA에서 지원을 인정 한다. S.C.H. 재생 의학 (RT3-07948)는 건강의 국가 학회 (U19 AI116484 및 R21 EB018407), 국립 과학 재단 (DMR 1508006), 및 캘리포니아 연구소에서 지원을 인정합니다. 이 연구는 유 니스 케네디 슈 라이버 국립 연구소의 아동 건강 및 인간 발달 (NICHD), 국립 연구소에 의해 지원 되는 재생 재활 연구 및 교육 (AR3T), 얼라이언스에서 자금 지원을 받은 신경 성 질환 및 치기 (NINDS), 국립 생물 의학 이미징 및 생명 공학 (NIBIB)의 수상 번호 P2CHD086843에서 건강의 국가 학회. 내용은 전적으로 저자의 책임 이며 반드시 국립 보건원의 의견을 대표 하지 않는다.

<ol>
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저자는 공개 없다.

재조합 형 단백질 표정과 정화 높은 재현성 바이오 소재를 합성 하는 강력한 도구입니다. 때문에 크게 상용화 분자 클로닝의 출현, 사용자 정의 재조합 형 플라스 미드 구입하실 수 있습니다 여러 공급 업체에서 크게 ELPs 같은 재료와 함께 일 하는 시간을 감소 시키는. 마찬가지로, plasmids 원래 작품 연방 계약에 의해 지원 되었다 미래 일 비영리 사용 될 것입니다 때 원래 실험실에서 직접 요청할 ?...

지난 세기 동안 2 차원 (2D) 조직 문화는 근본적인 세포 생물학에서 공부 시험관에대 한 통합 도구 집합으로 개발 했다. 또한, 2 차원 세포 배양에 대 한 상대적으로 저렴 하 고 간단한 프로토콜은 많은 생물학과 의학 분야에 걸쳐 그것의 채용에 이르렀다. 그러나, 과거의 연구 표시 했다 전통적인 2D 플랫폼 이어질 수 있는 결과 그 수집 된 vivo에서에서 현저 하 게 이탈, 귀중 한 시간을 일으키는 원인이 되 고 자금 낭비 임상 지향적인된 연구1,2, 3. 우리와 다른 가설 것이 불일치 최적의 확산 및 다양 한 종류의 세포 성숙에 필요한 수 있는 2D 표면에 경작 하는 셀에 제공 하는 네이티브 생화학 및 생물 단서의 부족에 표시 될 수 있습니다.
이러한 제한 및 도움 다리 2D 간의 격차를 해결 하기 위해 생체 외에서 그리고 vivo에서 학문, 연구원은 세포 캡슐화1,4,5 개발된 3 차원 (3D) 히드로 플랫폼을가지고 ,6. Hydrogels는 이상적인 재료는 세포 외 기질 (ECM) 생체 내에서 그들의 조직 같은 기계적 성질 및 영양분의 빠른 수송을 가능 하 게 물을 부 어 구조의 내 인 성 microenvironment을 정리 하 고 신호7,8요인. 또한, 3D hydrogels 독립 제어할 발판의 기계 및 생 화 확 적인 속성을 설계할 수 있습니다. 매트릭스 역학9,10,,1112 와 셀 접착제 ligands13,,1415 셀에 영향을 잘 알려져 있습니다. 생체 외에서 행동 하 고 vivo에서. 따라서, 가변 속성 3D hydrogels 셀과 그들의 microenvironment 간의 인과 관계를 연구 하는 플랫폼을 제공 합니다. 이상적인 3D 하이드로 겔 매트릭스에 대 한 기준 간단 하 고, 비 세포 독성 세포 캡슐화로 생리 적으로 관련 된 기계적 특성의 독립적인 tunability 및 기본 세포 접착 모티프의 모방을 포함.
모두 합성 (예., 폴 리 에틸렌 글리콜, polylactic 산, 폴 리 (glycolic acid)) 자연스럽 게 파생 (예., alginate, 콜라겐, Matrigel) hydrogels 있다 이점이 문화 플랫폼 2D 생체 외에서 ; 그러나, 그들은 또한 그들의 적용을 제한 하는 중요 한 단점이 있다. 첫 번째, 포유류 세포에 잠재적으로 독성이 될 수 있는 가혹한 가교 조건을 요구 하는 많은 합성 하 고 자연스럽 게 파생 된 플랫폼, 선도적인 세포 생존7감소. 또한, 많은 합성 플랫폼 네이티브 bioactivity 부족 그리고 증가 비용 및 복잡성16을 추가할 수 있는 보조 화학 반응을 통해 공업화 될 해야 합니다. 마지막으로, 자연스럽 게 파생 된 자료는 일반적으로 내장 바이오 액티브 도메인을 포함, 그들은 종종 높은 일괄 배치 변화를 시달려 고 종종 상대적으로 약한 젤7,17형성에 제한 됩니다.
재조합 단백질 공학 단백질 시퀀스를 명시적으로 제어 함으로써 재료 설계에 대 한 고유한 도구 집합을 제공 하 고, 확장 하 여, 최종 하이드로 겔의 잠재적인 기계 및 생 화 확 적인 재산18발판. 또한, 대장균 (대장균) 단백질을 표현 하의 잘 알려진 생물 학적 기계를 활용 하 여 재료 생산 수 있습니다 효율적이 고 일관 되 게 제한 남북 및 내부-일괄 가변성. 여기에 제시 된 엘라 스 틴 같은 단백질 (ELP)는 3 개의 조작된 도메인: 항 체를 태그를 통해 붙일 라벨을 허용 (1) T7, His6 태그, 수 여 탄성 기계적 성질 및 화학에 대 한 (2)는 ' 엘라 스 틴 같은 ' 지역 가교, (3) 세포 접착 모티프에 대 한 인코딩 ' 바이오-액티브 ' 지역.
엘라 스 틴 같은 지역 4 'Xaa' 아미노산 사이트 이소류신 (Ile), 하지만 될 수 있는 어떤 아미노산 프롤린 제외 될 하도록 정식 (발-프로-Gly-Xaa-Gly)5 엘라 스 틴 시퀀스를 기반으로 합니다. 이 시퀀스는 간단한 정화 후 식 열 사이클링19,20를 통해 악용 될 수 있는 낮은 중요 한 솔루션 온도 (LCST) 동작으로 재조합 ELPs endows. 이 LCST 속성 열 집계에 다른 온도에 게스트 'Xaa' 찌 꺼 기21,22를 수정 하 여 조정 될 수 있습니다.
여기, 5 엘라 스 틴 같은 반복 중 하나에 'Xaa' 위치는 아민 제시 리 (리스) 아미노산, 히드로 가교에 이용 되는 대체 되었습니다. 우리의 이전 작품 비 세포 독성과 강력한 아민 반응 crosslinker tetrakis (hydroxymethyl) phosphonium 염화 (THPC)23반응을 통해 가교를 보이고 있다. 다양 한 전체 단백질 콘텐츠 및 crosslinker 농도, 우리 hydrogels 순수 관련 강성 범위 (~0.5-50 kPa)9,,2324스팬 조정 될 수 있습니다 생산 수 있습니다. 기계적 성질, 튜닝 외에 히드로 내 세포 접착 ELP 단백질의 백본 내의 정식 셀 접착제 도메인의 통합에서 유래한 다. 예를 들어 비 바인딩 'RDGS' 변종 세포-매트릭스 접착24을 제한 확장된 fibronectin 파생 'RGDS' 아미노산 시퀀스의 세포 접착 및 스크램블, 동안 구조적 유연성을 허용 합니다. 세포 접착 비 접착 단백질 뿐만 아니라 총 단백질 농도의 비율을 변조, 우리가 효과적으로 리간드 농도의 넓은 범위에 걸쳐 hydrogels 생산 수 있습니다. 릴리스할, 히드로 플랫폼 독립적으로 튜닝할 수 없는 다양 한 종류의 세포 최적의 3D 문화에 대 한 분리 된 생 화 확 및 생물 속성을 개발 했습니다.
매트릭스 강성 및 접착제 ligand tunability, 재조합 hydrogels 셀 확산, 확산, 및4 3D 컨텍스트 내에서 마이그레이션에 필요한 디자인 특정 소재 저하 프로필 기능 제공 , 9.이 저하 특히 대상으로 확장된 'RGDS'9 또는 엘라 스 틴 같은 시퀀스25프로 테아의 분 비 세포에서 제공. ELP hydrogels 세포 생존 능력 및 기능 immunocytochemistry 양적 역에 대 한 DNA/RNA/단백질 추출 등 공부에 필요한 후속 생 화 확 적인 분석 실험을 지원 하기 위해 표시 되었습니다 또한 전사-연쇄 반응 (qRT-PCR)와 서 부 오9점. ELP 변종 vivo에서 모델의 수에도 사용 되었습니다 그리고26면역 시스템 의해 잘 용납 될 것으로 알려져 있습니다.
합쳐, ELP 다양 한 합성 또는 자연스럽 게 파생 된 소재 플랫폼, 생화학 및 생물 tunability의 동일한 정도 부족에 비해 혜택을 자랑 하는 셀 캡슐화 연구 소재 플랫폼으로 고 재현성입니다. 또한, ELP의 간단 하 고 세포 독성 비 사용 다양 한 세포 유형 (예., 병아리 지 루트 중추14,24, murine 신경 조상 세포9, 인간 간 엽 줄기 세포27, 소 신생아 chondrocytes28, 인간 내 피 세포29,30) 2 차원 세포 배양에 비해 생 3D ECM의 더 순수 관련 모델에 대 한 수 있습니다. 여기, 우리 recombinantly 파생의 표현에 대 한 프로토콜을 제시, 3D에 대 한 가변 히드로 플랫폼으로 사용 하기 위해 ELPs 세포 캡슐화. 우리는 더 다운 스트림 형광 라벨 및 캡슐화 된 세포의 confocal 현미경 검사 법에 대 한 방법론을 제시.

<table><tbody><tr><td>&lt;strong&gt;Elastin-Like Protein Expression and Purification&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 cm Petri Dishes</td><td>Thermo Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>70% Ethanol</td><td>RICCA Chemical</td><td>2546.70-1</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>Sigma-Aldrich</td><td>A3920-500G</td><td></td></tr><tr><td>Ampicillin</td><td>Thermo Fisher Scientific</td><td>BP1760-25G</td><td></td></tr><tr><td>Bacto Agar</td><td>Thermo Fisher Scientific</td><td>9002-18-0&amp;nbsp;</td><td></td></tr><tr><td>BL21(DE3)pLysS Competent Cells</td><td>Invitrogen</td><td>C606003</td><td></td></tr><tr><td>Chloramphenicol</td><td>Amresco</td><td>0230-100G&amp;nbsp;</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>EDTA disodium salt, dihydrate</td><td>Thermo Fisher Scientific</td><td>O2793-500</td><td></td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific</td><td>BP229-4</td><td></td></tr><tr><td>Isopropanol</td><td>Thermo Fisher Scientific</td><td>A451-4</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>BP1755-10G</td><td></td></tr><tr><td>Luria Broth</td><td>EMD Millipore</td><td>1.10285.5007</td><td></td></tr><tr><td>Parafilm</td><td>VWR</td><td>52858-000</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>MP Biomedicals</td><td>195381</td><td></td></tr><tr><td>Sodium Chloride</td><td>Thermo Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S 8045-1KG</td><td></td></tr><tr><td>Syringe Filter Unit (0.22 &amp;mu;m)</td><td>Millipore</td><td>SLGP033RB</td><td></td></tr><tr><td>Terrific Broth</td><td>Millipore</td><td>71754-4</td><td></td></tr><tr><td>Tris Base</td><td>Thermo Fisher Scientific</td><td>BP152-1</td><td></td></tr><tr><td>&lt;strong&gt;Cell Encapsulation in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.22 &amp;mu;m syringe filters</td><td>Millipore</td><td>SLGV004SL</td><td></td></tr><tr><td>0.5 mm thick silicone sheet</td><td>Electron Microscopy Science</td><td>70338-05</td><td></td></tr><tr><td>24-well tissue culture plates&amp;nbsp;</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>Disposable Biopsy Punch (2 mm)</td><td>Integra Miltex</td><td>33-31</td><td></td></tr><tr><td>Disposable Biopsy Punch (4 mm)</td><td>Integra Miltex</td><td>33-34</td><td></td></tr><tr><td>Disposable Biopsy Punch (5 mm)</td><td>Integra Miltex</td><td>33-35</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s phosphate buffered saline (DPBS)&amp;nbsp;</td><td>Corning</td><td>21-031-CM</td><td></td></tr><tr><td>No. 1 12 mm glass coverslips</td><td>Thermo Fisher Scientific</td><td>12-545-80</td><td></td></tr><tr><td>Tetrakis(hydroxymethyl)phosphonium chloride (THPC)</td><td>Sigma-Aldrich</td><td>404861-100ML</td><td></td></tr><tr><td>0.5% Tryspin/EDTA</td><td>Thermo Fisher&amp;nbsp;</td><td>15400054</td><td></td></tr><tr><td>&lt;strong&gt;Immunocytochemistry of Cells in 3D ELP Hydrogels&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>16% (w/v) Paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15701</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Roche</td><td>3116956001</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Molecular Probes</td><td>D1306&amp;nbsp;</td><td></td></tr><tr><td>Donkey Serum</td><td>Lampire Biological Labs</td><td>7332100</td><td></td></tr><tr><td>Goat anti-mouse Secondary Antibody (AF488)</td><td>Molecular Probes</td><td>A-11017</td><td></td></tr><tr><td>Goat anti-rabbit Secondary Antibody (AF546)</td><td>Molecular Probes</td><td>A-11071</td><td></td></tr><tr><td>Goat Serum</td><td>Gibco</td><td>16210-072</td><td></td></tr><tr><td>Mouse Nestin Primary Antibody</td><td>BD Pharmingen</td><td>556309</td><td></td></tr><tr><td>Mouse Sox2 Primary Antibody</td><td>Cell Signaling Technology</td><td>23064S</td><td></td></tr><tr><td>Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>Vectashield Hardset Mounting Medium&amp;nbsp;</td><td>Vector Labs</td><td>H-1400&amp;nbsp;</td><td></td></tr></tbody></table>

production of elastin-like protein hydrogels for encapsulation and immunostaining of cells in 3d

2 차원 (2D) 조직 문화 기술 근본적인 세포 생물학의 우리의 이해에 필수적인 되었습니다. 그러나, 전통적인 2D 조직 문화 시스템 부족 결과 사이의 중요 한 결과 3 차원 (3D) 매트릭스 분리 수집 생체 외에서 그리고 vivo에서. 이 한계를 해결 하기 위해 연구자는 vivo에서 세포 microenvironment의 생 화 확 및 생물 속성을 흉내낼 수 있는 3 차원 하이드로 겔 조직 문화 플랫폼 설계 있다. 이 연구는 3D 셀 캡슐화 및 다운스트림 생 화 확 적인 분석 실험을 지 원하는 소재 플랫폼을 개발할 필요가 동기가 있다. 재조합 단백질 공학 단백질 시퀀스의 특정 컨트롤에 대 한 허용 및 따라서, 확장, 결과의 잠재적인 기계 및 생 화 확 적인 속성 3D 하이드로 겔 소재 설계 및 개발에 대 한 고유한 도구 제공 매트릭스입니다. 여기, 우리는 recombinantly 파생 된 엘라 스 틴 같은 단백질 (ELP), 양식 hydrogels 독립적으로 조정할 수 있는 기계적 특성 및 세포 접착 ligand 농도에 사용 될 수 있는 표현에 대 한 프로토콜을 제시. 우리 더 ELP hydrogels 내 셀 캡슐화에 대 한 방법론을 제시 하 고 다운스트림 분석 및 정량화에 대 한 셀을 포함의 후속 immunofluorescent 얼룩.

ELPs이이 프로토콜에 사용 되는 5 개 영역으로 구성: T7 태그, His6 태그, enterokinase (EK) 분열 사이트, 생체 활성 영역 및 엘라 스 틴 같은 지역 (그림 1). T7 His6 태그 표준 서쪽 오 점 기술을 통해 쉽게 식별할 수 있습니다. EK 분열 사이트의 소개 태그 영역의 효소 제거 필요한 경우 수 있습니다. 바이오 액티브 지역 확장, fibronectin 파생 셀-접착제 ('RGDS') 또는 ...

Watch this Scientific Journal Video about Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D at JoVE.com

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

그들은 완전 한 tunability 폴리머 백본 및 따라서 셀 microenvironment의 허용으로 재조합 단백질 엔지니어링 hydrogels는 3D 세포 배양에 대 한 유리한 있습니다. 여기, 우리 재조합 엘라 스 틴 같은 단백질 정화의 과정 및 3 차원 하이드로 겔 셀 캡슐화에의 응용을 설명합니다.

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 ...

production-elastin-like-protein-hydrogels-for-encapsulation

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.3K 조회수.  Stanford University. 그들은 완전 한 tunability 폴리머 백본 및 따라서 셀 microenvironment의 허용으로 재조합 단백질 엔지니어링 hydrogels는 3D 세포 배양에 대 한 유리한 있습니다. 여기, 우리 재조합 엘라 스 틴 같은 단백질 정화의 과정 및 3 차원 하이드로 겔 셀 캡슐화에의 응용을 설명합니다.

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

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.

셀 문화 응용 프로그램에 대 한 단백질 기반 Hydrogels 아키텍처의 쉬운 조작

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

연구

생화학

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

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

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

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

단계 성장 PEG-펩타이드 Hydrogels에서 β-세포 Spheroids의 생성 및 복구

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

생체공학

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

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

조직 공학을위한 3D Hydrogels에서 셀룰러 봉지

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

생물학

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

세포 외 매트릭스 내에서 임베디드 공학 3 차원 상피 조직

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

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 ...

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.

주 사용 재생 치료와 높은 처리량 마약 검사에 대 한 3D Microtissues

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

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

부유 된 3D 바이오 프린팅을위한 겔화 동안 전단 처리에 의해 형성된 Agarose 유체 겔

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...

ECM 경직에 대한 세포 반응을 프로브하기 위한 광조정 가능한 바이오프린팅 하이드로겔

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.

저자 스포트라이트: 3D 프린팅 광조정 가능 하이드로겔을 사용한 만성 폐 질환 이해 

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

섬유아세포 활성화를 연구하기 위한 3D 바이오프린팅 광조정 가능 하이드로겔

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