1. 기본 관절 Chondrocytes의 절연 동물 관절의 관절 표면에서 수확 10-15 전체 두께 연골의 조각 (로컬 abbatoir에서 얻은 skeletally 성숙 소의 손바닥 뼈 - phalangeal 공동을 지정할 수). <ol><li> 37 번 2 시간에 대한 햄의 F-12 (w / V)에서 0.5 % 프로테아제의 20 ML에 100mm 페트리 접시 및 배양 ° C.의 장소 연골 슬라이스 햄의 F-12 문화 미디어에 세 번 헹구고, 0.15 %의 Collagenase의 20 ML을 품다 37 번 밤새 햄의 F-12 문화 미디어 ° C.에 </li><li> 깨끗한 그릇에 200 메쉬 스크린 필터를 통해 세포 현탁액을 필터링 할 수 있습니다. 햄의 F-12의 5 ML과 필터를 깨끗이 씻어 요리에 추가 할 수 있습니다. 50 ML 원뿔 튜브에 세포 현탁액을 전송합니다. 햄의 F-12의 10 ML과 페트리 접시를 씻어과 원뿔 튜브에 추가 할 수 있습니다. </li><li> 실온에서 6-8 분 동안 600-800 rcf에있는 튜브를 원심 분리기. </li><li> 일을 방해하지 보장 표면에 뜨는을 대기음전자 세포 펠렛과 햄의 F-12 문화 미디어 40 ML에 다시 - 일시 중지합니다. </li><li> 6-8 분에 600-800 rcf에있는 튜브를 원심 분리기. </li><li> 3 회 총에 대한 단계 1.4 1.5 두 번 더 반복합니다. 전에 세 번째 시간 centrifuging에, 교반을 통해 펠릿을 resuspend와 셀 계산을위한 500 μl 나누어지는을 구하십시오. </li><li> hemacytometer와 역 가벼운 현미경으로 Trypan 파랑 제외 방법 9를 사용 가능한 셀을 계산합니다. </li><li> 표면에 뜨는을 대기음하고 원하는 심는 밀도를 두 배로 햄의 F-12의 미디어 볼륨에서 펠렛을 다시 일시 중지 (예 : 20 × 10 10 × 10 6 세포 / 캡슐화 한 후 ML의 최종 농도 6 셀 / ML). </li></ol> 2. Chondrocyte - 아가로 오스의 히드로 겔 캡슐화 <ol><li> 열 0.8 g autoclaved 유형 VII 아가로 오스 용해 ° C까지 120 세트 핫 플레이트에 살균 1X PBS (산도 7.4)의 20 ML 인치 일단 용해, ° C보다 높은 온도로 60 열을 낮출 wil난 세포 생존에 영향을 미칩니다. </li><li> 50 ML 원뿔 튜브에서 철저하게 아가로 오스 (최종 2퍼센트 아가로 오스의 히드로 겔에 대한 예 4퍼센트 아가로 오스 정지)의 두 원하는 농도로 세포 현탁액의 동등한 볼륨을 섞는다. 그들은 세포 생존에 영향을 미치는 피로 수명을 만들 수 있으므로 큰 거품의 생성을 피하십시오. </li><li> 조심스럽게 나누어지는 10 60mm 직경 페트리 접시에 chondrocyte - 아가로 오스 혼합물의 ML, 큰 거품의 생성을 피하면서. </li><li> gelled 때까지 실온에서 30 분에 서 보자. </li><li> 많은 chondrocyte - 아가로 오스의 히드로 겔 샘플로 4mm 생검 펀치를 사용하여 (일반적으로 20-30) 필요에 따라 얻습니다. </li><li> 마이크로 미터를 사용하여 대표 시료의 물리적 인 크기를 (직경 높이) 측정하고 기록합니다. 구조는 샘플 높이 차이가 큰 2 %였다 지역에서 얻은 샘플을 폐기, 높이 약 5mm해야합니다. </li><li> 새로운 60mm 페트리 접시와 보충에 샘플을 전송전체 미디어의 10 ML (20 MM HEPES, 100 μg / ML 아스코르브 산, 그리고 배 항생제 / antimycotics와 햄의 F-12 문화 미디어 등 20% 태아 소 혈청)과. </li></ol> 3. Chondrocyte - 아가로 오스 Hydrogels의 기계 자극 및 Radiolabelling <ol><li> 압축 장비의 금속 구성 요소를 압력솥. 70 % 에탄올 (야간) 및 UV 빛 (적어도 15 분 용)과 플라스틱 구성 요소를 살균. </li><li> 24 잘 문화 판의 우물에서 장소 12 멸균 플라스틱 유지 링 (구조 직경보다 큰 내부 직경) (N = 6, 샘플 및 컨트롤을), 이상 떨어지는 집게를 사용 구조를 유지합니다. </li><li> 집게를 사용 조심스럽게 유지 링에 각각 확보, 페트리 접시에서 24 잘 판에 chondrocyte - 아가로 오스의 hydrogels를 전송할 수 있습니다. </li><li> 전체 미디어 400 μl (예를 들면 20% 20 MM HEPES, 100 μg / ML의 아스코르브 산으로 햄의 F-12 문화 미디어 FBS 및 전송잘 각 샘플에) 항생제 / antimycotics 2 배. </li><li> 살균 압축 장비 (그림 1) 조립 및 세트 나사와 platens을 확보. 24 잘 문화 판에 압축 장비를 연결합니다. </li><li> 플래 튼 - 히드로 겔 연락처 (제로 변형 상태)를 구축하기 위해 세트 나사를 해제하고 다시 고정하십시오. </li><li> 마하-1 Micromechanical 테스트 시스템으로 조립 압축 장비를로드합니다. 세트 나사와 장소에서 위치 핀과 잠금을 통해 수직 단계에 보안 장비. 간격 지그를 제거합니다. </li><li> 주파수 (예 : 1 Hz에서) 및 기간 또는 사이클의 수 (60 분에 예를 들어 시간 간격까지), 원하는 진폭 (샘플의 원래 높이를 기준으로 예를 들어 10 %의 변형 진폭)에서 동적 압축 하중을 적용합니다. </li><li> 기계 자극 완료되면, 간격 지그를 대체 위치 핀 세트 나사를 풀어 마하-1 Micromechanical 테스트 시스템에서 압축 장비 및 문화 판을 제거합니다. </lI&gt; <li> Radiolabel 셀 - 히드로 겔 원하는 동위 원소 (chondrocyte 특정 콜라겐 합성과 [35 S] - 유황 proteoglycan 합성을위한에 대한 예 [3 H]-프롤린 단백질 라벨) 5 μCi으로 미디어를 보완하여 구조. </li><li> 37에서 24 시간에 대한 방사성 동위 원소 표지 된 구조를 길러 ° C, 5 % 2 10 CO. </li><li> 방사성 동위 원소 표지 된 히드로 겔 구조를 수확하고 비법 인 동위 원소를 제거 할 1X PBS (산도 7.4)에서 3 번 씻어. papain (20 MM의 암모늄 아세테이트 40 μg / ML, 65에서 1 ㎜ ethyldiaminetetraacetic 산 2 밀리미터 dithiothreitol ° C 72 시간 용)를 사용하여 다이제스트 샘플. </li><li> PicoGreen 염료 분석 11, 법인의 동위 원소 활동, 12,13 즉시 다음 papain을 소화을 계산 β-액체 섬광에 의해 DNA의 콘텐츠에 대한 표준화를 사용하여 DNA 콘텐츠에 대한 분석 요약. </li></ol> 참고 : 방사성 물질의 구입 및 처분 (폐기물 동위 원소 및 항목연락처 방사성 물질에 와서 도움이 될 수 있도록 () 및 / 또는 정부) 정책과 방사성 물질의 안전한 취급 및 폐기 절차 관련 기관을 준수해야합니다. 4. 대표 결과 소 관절 chondrocyte - 아가로 오스 hydrogels 구조 (10 × 10 6 세포 / ML 캡슐화 된 셀과 2퍼센트 아가로 오스)는 기계적으로 20-60 분 (또는 1200에 3600 사이클 1 Hz의 주파수 10 % 압축 응력의 진폭에 자극했다 ) 및 방사성 동위 원소의 설립에 의한 DNA 콘텐츠 및 세포 외 기질 합성에 assayed. DNA와 세포 외 기질 합성은 복용에 의존 방식으로 영향을했습니다. 자극의 60 분 (그림 2) 아무런 영향을 전시하지 반면, DNA 내용은, 자극의 20 ~ 30 분 (P &lt;0.01)의 결과로 상당한 35 % 감소 전시. 연골 특정 콜라겐과 proteoglycan 합성 ([3 H]-프롤린 및 결정각각 [35 S] - 유황 설립은) 전혀 관찰 할 수있는 효과를 전시하지 자극 60 분 (그림 3)과 자극을 20 ~ 30 분 (P &lt;0.01)에 대한 응답으로 약 60 %의 상당한 증가를 전시. <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="그림 1" /> . chondrocyte - 아가로 오스 구조를 촉진을위한 맞춤식 동적 압축 장비의 그림 1 도식 왼쪽 :. 조립 장비 마우스 오른쪽 :. 개별 구성 요소를 보여주는 뷰를 폭발했다. <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="그림 2" /> chondrocyte - 아가로 오스의 DNA 콘텐츠에 그림 2. 변경은 기계적으로 20-60 분 1 Hz에서 10 % 압축 변형 진폭 (± SEM, N = 6/group을 의미)에서 자극 hydrogels. chondrocyte - 아가로 오스의 세포 외 기질 합성 (콜라겐과 proteoglycans)의 그림 3. 변경은 기계적으로 20-60 분 (± SEM, N = 6/group을 의미) 1 Hz에서 10 % 압축 변형 진폭에 따라 자극 hydrogels.

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관심 없음 충돌이 선언 없습니다.

<html> 셀 - 시드 아가로 오스 hydrogels로 제어 기계 자극을 적용하기위한 설명 방법은 chondrocyte 신진 대사에 동적 압축 힘의 효과에 직접 조사 할 수 있습니다. 유지 링과 함께 맞춤형 테스트 장비의 사용은 샘플 팁의 잠재적 인 문제를 방지하기 위해 구조에 대한 측면 제약 조건을 제공했습니다. 세트 요원 확보 사망 가중 로딩 platens의 사용은 샘플의 높이에 잠재적 인 차이에도 불구하고 구조와 직접?...

<table border="1"><tbody><tr><td> 시약 또는 장비의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 햄의 F-12 </td><td> 온도 피셔 과학 </td><td> SH3001002 </td><td></td></tr><tr><td> Collagenase </td><td> 시그마 알드리치 (주) </td><td> C0130 </td><td></td></tr><tr><td> 단백 분해 효소 </td><td> 시그마 알드리치 (주) </td><td> P5147 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> 시그마 알드리치 (주) </td><td> F1051 </td><td></td></tr><tr><td> 아스 코브 산 </td><td> 시그마 알드리치 (주) </td><td> A4034 </td><td></td></tr><tr><td> 항생제 / antimycotics </td><td> 시그마 알드리치 (주) </td><td> A5955 </td><td></td></tr><tr><td> HEPES </td><td> Bioshop 캐나다 (주) </td><td> HEP001 </td><td></td></tr><tr><td> Trypan 파랑 </td><td> 시그마 알드리치 (주) </td><td> 93,595 </td><td></td></tr><tr><td> 라이 밝은 줄 Hemacytometer </td><td> Hausser 과학 </td><td> 1490 </td><td></td></tr><tr><td> 양의 - IT PicoGreen </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> 파파야 라텍스에서 Papain </td><td> 시그마 알드리치 (주) </td><td> P3125 </td><td></td></tr><tr><td> 암모니아 아세테이트 </td><td> 시그마 알드리치 (주) </td><td> A1542 </td><td></td></tr><tr><td> Ethyldiaminetetraacetic 산 </td><td> 시그마 알드리치 (주) </td><td> E9884 </td><td></td></tr><tr><td> DL-Dithiothreitol </td><td> 시그마 알드리치 (주) </td><td> 43,819 </td><td></td></tr><tr><td> 낮은 녹는 점 아가로 오스, 유형 VII </td><td> 시그마 알드리치 (주) </td><td> A9045 </td><td></td></tr> &lt;TR&gt; <td> 메쉬 스크린 (200) 필터 </td><td> 시그마 알드리치 (주) </td><td> S4145 </td><td></td></tr><tr><td> 마하-1 Micromechanical 시험기 </td><td> Biomomentum 주식회사 </td><td> V500cs </td><td></td></tr><tr><td> 압축 로딩 파티는 </td><td> 주문품의 </td><td></td><td> 비슷한 제품은 Biomomentum 주식회사에서 제공 될 수 </td></tr><tr><td> 매 24 자 문화 플레이트 </td><td> 온도 피셔 과학 </td><td> B353047 </td><td></td></tr><tr><td> β-액체 신틸레이션 계수기 </td><td> Beckman 보습 바로 앞에 달린 풀 베는 날 </td><td> LS6500 </td><td></td></tr><tr><td> [3H] 프롤린 </td><td> Perkin-엘머 </td><td> NET323005MC </td><td></td></tr><tr><td> [35S] 황 </td><td> Perkin-엘머 </td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

기계 모욕이나 관절염과 같은 질환에 의해 저하에 의해 손상되면 관절 연골은 제한된 복구 능력을 앓고. 이 결핍을 해결하려면 몇 가지 의료 개입이 개발되었습니다. 하나의 방법은 조직 - 엔지니어링 연골과 손상된 영역을 표면화하는 것입니다, 그러나, 엔지니어링 조직은 일반적으로 장기 survivability에 의문을 제기하고, 기본 연골의 생화학 특성과 내구성이 부족합니다. 이 주입 된 자료를 보호하기 위해 주변 조직에 의존, 작은 초점 결함의 수리에 연골 조직 공학의 응용 프로그램을 제한합니다. 개발 조직의 속성을 개선하기 위해 기계 자극은 연골 성의 세포 외 기질의 합성뿐만 아니라 엔지니어링 조직의 결과 기계적 성질을 향상시키기 위해 활용 인기있는 방법입니다. 기계 자극은 조직에 힘을 적용 생체에 이러한 경험과 유사한 구성합니다. 이기계 환경, 부분적으로, 기본 조직 1,2의 개발과 유지 보수를 조절하는 전제에 기반을두고 있습니다. 연골 조직 공학에서 기계 자극의 가장 일반적으로 적용되는 형태는 1 Hz에서 1,3의 주파수에서 약 5~20%의 physiologic 변종에 동적 압축합니다. 몇몇 연구는 동적 압축의 효과를 조사했고 최종적으로 개발 된 조직 4-8의 기능적 특성에 영향을 미치는, chondrocyte 신진 대사와 생합성에 긍정적 인 효과를 보여 주었다. 이 논문에서, 우리는 기계적으로 동적 압축에 따라 chondrocyte - 아가로 오스의 히드로 겔 구조를 자극하고 생화학 및 방사성 동위 원소 assays을 통해 생합성의 변화를 분석 할 수있는 방법을 보여줍니다. 이 방법은 쉽게 기계적 자극의 결과로 세포의 반응에 잠재적으로 유도 변경 사항을 평가하기 위해 수정할 수 있습니다.

Watch this Scientific Journal Video about Mechanical Stimulation of Chondrocyte-agarose Hydrogels at JoVE.com

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

chondrocytes의 연골 성의 세포 외 기질의 생합성은 기계적 자극의 응용 프로그램에 의해 영향을받을 수 있습니다. 이 방법은 3D 구조와 chondrocyte 신진 대사를 유도 변화의 평가에 캡슐화 chondrocytes에 동적 압축 변종을 적용하는 기술에 대해 설명합니다.

Chondrocyte - 아가로 오스 Hydrogels의 기계적 자극

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

mechanical-stimulation-of-chondrocyte-agarose-hydrogels

Research

JoVE Journal

Biology

11.6K 조회수.  Queen's University. chondrocytes의 연골 성의 세포 외 기질의 생합성은 기계적 자극의 응용 프로그램에 의해 영향을받을 수 있습니다. 이 방법은 3D 구조와 chondrocyte 신진 대사를 유도 변화의 평가에 캡슐화 chondrocytes에 동적 압축 변종을 적용하는 기술에 대해 설명합니다.

비디오: Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

텐든의 전 생체내 기계 중

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

연구

생물학

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

순환 Uniaxial 스트레인을 사용하여 줄기 세포의 기계적 자극

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

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 ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

This paper aims to instruct the reader in the operation of an integrated atomic force-optical imaging microscope for mechanical stimulation of live cells in culture. A step-by-step protocol is presented. A representative data set that shows live cell response to mechanical stimulation is presented.

기계 자극에 라이브 세포 응답 통합 광학과 원자 힘 현미경에 의해 연구

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

세포 단일 층의 기계적 자극에 의​​한 칼슘 웨이브 전파 : 소 각막 내피 세포의 예

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, the effect of tissue elasticity on cell function is a major area of mechanobiology research because tissue stiffness modulates with disease, development, and injury. Static tissue-mimicking materials, or materials that cannot alter stiffness once cells are plated, are predominately used to investigate the effects of tissue stiffness on cell functions. While information gathered from static studies is valuable, these studies are not indicative of the dynamic nature of the cellular microenvironment in vivo. To better address the effects of dynamic stiffness on cell function, we developed a DNA-crosslinked polyacrylamide hydrogel system (DNA gels). Unlike other dynamic substrates, DNA gels have the ability to decrease or increase in stiffness after fabrication without stimuli. DNA gels consist of DNA crosslinks that are polymerized into a polyacrylamide backbone. Adding and removing crosslinks via delivery of single-stranded DNA allows temporal, spatial, and reversible control of gel elasticity. We have shown in previous reports that dynamic modulation of DNA gel elasticity influences fibroblast and neuron behavior. In this report and video, we provide a schematic that describes the DNA gel crosslinking mechanisms and step-by-step instructions on the preparation DNA gels.

Our laboratory has developed DNA-crosslinked polyacrylamide hydrogels, a dynamic hydrogel system, to better understand the effects of modulating tissue stiffness on cell function. Here, we provide schematics, descriptions, and protocols to prepare these hydrogels.

DNA-가교 폴리 아크릴 아미드 하이드로 겔의 제조

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

Multifunctional, Micropipette-based Method for Incorporation And Stimulation of Bacterial Mechanosensitive Ion Channels in Droplet Interface Bilayers

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. It has been discovered and rigorously studied using the patch-clamp technique for almost three decades. Its basic role of translating tension applied to the cell membrane into permeability response makes it a strong candidate to function as a mechanoelectrical transducer in artificial membrane-based biomolecular devices. Serving as building blocks to such devices, droplet interface bilayers (DIBs) can be used as a new platform for the incorporation and stimulation of MscL channels. Here, we describe a micropipette-based method to form DIBs and measure the activity of the incorporated MscL channels. This method consists of lipid-encased aqueous droplets anchored to the tips of two opposing (coaxially positioned) borosilicate glass micropipettes. When droplets are brought into contact, a lipid bilayer interface is formed. This technique offers control over the chemical composition and the size of each droplet, as well as the dimensions of the bilayer interface. Having one of the micropipettes attached to a harmonic piezoelectric actuator provides the ability to deliver a desired oscillatory stimulus. Through analysis of the shapes of the droplets during deformation, the tension created at the interface can be estimated. Using this technique, the first activity of MscL channels in a DIB system is reported. Besides MS channels, activities of other types of channels can be studied using this method, proving the multi-functionality of this platform. The method presented here enables the measurement of fundamental membrane properties, provides a greater control over the formation of symmetric and asymmetric membranes, and is an alternative way to stimulate and study mechanosensitive channels.

Bacterial mechanosensitive channels can be used as mechanoelectrical transducers in biomolecular devices. Droplet interface bilayers (DIBs), cell-inspired building blocks to such devices, represent new platforms to incorporate and stimulate mechanosensitive channels. Here, we demonstrate a new micropipette-based method of forming DIBs, allowing the study of mechanosensitive channels under mechanical stimulation.

다기능, 물방울 인터페이스 이중층에서 세균 Mechanosensitive 이온 채널의 설립과 자극에 대한 방법을 마이크로 피펫 기반

MscL, a large conductance mechanosensitive channel (MSC), is a ubiquitous osmolyte release valve that helps bacteria survive abrupt hypo-osmotic shocks. ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

3 차원 히드로 겔의 이미지를 FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Dictyostelium discoideum 응답 심각한 기계적인 자극의 평가

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

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In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...