The research leading to these results has received funding from the European Research Council under grant agreement ERC-2010-StG-260991.

체적 휴대용 광 음향 프로브를 동작하기위한 상세한 절차는 다음과 같다. 이 절차는 동물과 인간 실험에 대한 승인 기관의 규정에 따라 수행된다. 1. 시스템 준비 <ol><li> 출력 광 빔을 안정화 작업 전에 15 분 ~ 예열 기간 동안 레이저를 전환합니다. </li><li> 피부 (그림 1)과 접촉 분리 막으로 물 둘러싸는 부분을 놓습니다. NOTE : (피부 접촉) 솔 레이팅 막과 트랜스 듀서 (구형 탐침의 중심)의 최대 감도 영역 사이의 거리가 유효 촬상 깊이를 설정한다. </li><li> 펌프에 의해 소자 분리 멤브레인과 탈 이온수 변환기의 표면 사이의 약 100 ml의 전체 부피를 채운다. </li><li> 더 누수 나 기포가 prese 없는지 확인NT. 다르게는, 물의 재순환을 제공하여 기포를 피한다. </li><li> RT에서 실험을 수행하고,이 온도로 유지되는 결합 매체 (물)을 보장한다. </li></ol> 2. 이미징 준비 <ol><li> 인간 이미징 준비. </li><li> 이미지에 바람직하지 않은 배경을 피하기 위해 제모 로션 투 - 이미징 될 부분으로부터 모발을 제거 (이 단계는 선택적이다). </li><li> 영역 주위 피부 효율적인 음향 결합을 제공하기 위해서 이미지화되도록 상 초음파 겔을 적용한다. 관심 영역에서 광 음향 프로브를 놓습니다. 기포가 초음파 커플 링 젤에 존재하지 않는 것을 확인합니다. </li><li> 동물 이미징 준비. </li><li> 동물 관리 및 실험 절차는 기관 및 정부 규칙 및 규정과 일치 있는지 확인합니다. </li><li> 이 지역에서 동물의 모피를 제거는 면도 로션으로 이미지화한다. 과 동물의 눈을 보호강렬한 펄스 레이저 방사선에 노출 건조와 손상을 방지 수의사 연고,. </li><li> 0.9 리터와 케타민 / 자일 라진 (xylazine) 체적 (100 ㎎ / kg KG 케타민 + 5 ㎎ / kg KG 크 실라) 실험 전에 또는 사용 이소 플루 란 마취 (2-3% ()의 복강 내 주사 (IP)을 사용하여 동물을 마취 / 실험 도중 최소 가스 흐름). 동물의 뒷다리의 반사를 확인하여 마취를 확인합니다. </li><li> 영역 주위의 피부에 초음파 겔 적용은 효율적인 음향 결합을 제공하고, 관심 영역의 광 음향 프로브를 배치하기 위해 영상화한다. 기포가 초음파 커플 링 젤에 존재하지 않는 것을 확인합니다. </li></ol> 3. 미리보기 동작 모드 <ol><li> 690 nm 내지 900 nm이고, 10 및 50 Hz에서의 펄스 반복 레이트의 촬상 파장 (들)을 수립. 음향 데이터 수집 시스템을위한 매개 변수를 선택 - 1 MΩ을입력 임피던스. 제 12 비트 수직 해상도 당 40 메가 샘플의 샘플링 레이트로 각각의 레이저 펄스를위한 2030 샘플들을 획득. 레이저의 Q 스위치 출력 인수를 트리거합니다. </li><li> 연산자와 환자 모두가 광 여기 파장 (들)에 적응 보호 고글을 사용해야합니다. 인간 실험 19 안전 노출 제한을 만족하고 동물에서 열 변형하고 피부 손상을 방지하기 위하여 조직 표면에서의 광의 조사량은 근적외선 파장 용 실험 기간 동안 20 엠제이 / cm 2 이하로 유지되도록 레이저 파워를 설정 . </li><li> 레이저의 펄스 반복 레이트에 대응하는 프레임 레이트에서 3 차원 영상을 시각화 할 수 있도록 처리 알고리즘의 GPU 구현을 미리보기 소프트웨어를 시작. </li><li> 프로브를 이동 및 / 또는 객체 시각화 성능을 최적화하고 관심있는 구조를 지역화하기 위해서 묘화 될. </li></ol> 4. 데이터 수집 <ol><li> 스캐닝 (휴대용) 모드에 대한 데이터 수집. </li><li> 필요한 경우, 관심 영역의 콘트라스트를 풍부하게 취득하기 전에 조영제를 주입. 참고 : 우리의 실험에서 우리는 조영 증강 인간의 이미징을 수행하지 않았습니다. 그러나, 다양한 조영제 잠재적이 목적을 위해 사용될 수있다. 인도 사이 아닌 그린 (ICG)는 성인 체중의 2 ㎎ / kg의 최대 권장량에서 콘트라스트 개선을 위해 사용될 수있는 임상 적으로 승인 광 조영제의 일례이다. </li><li> 미리 소프트웨어의 실행을 유지하는 3.1에서 설명한 파라미터들에 데이터 수집을위한 하드웨어 시작. 부드럽게 관심 구조를 추적하는 묘화 영역의 주위에 탐침을 이동. NOTE는 : 복수의 레이저 파장에서의 이미지를 동시에 획득되는 경우, 휴대용 프로브 모드에서 동작의 속도가 현저하게 감소되어야한다 (바람직하게는 아래 2mm / 초의스펙트럼 섞이지 이미지 모션 관련 아티팩트를 회피하기 위해 50 Hz의 레이저 펄스 반복율) 미국. </li><li> 정지 모드에 대한 데이터 수집. </li><li> 홀더 상으로 묘화 객체 (예., 동물)와 휴대용 프로브를 탑재하고 미리보기 소프트웨어의 실행을 유지하는 3.1에서 설명한 매개 변수 획득을 시작. </li><li> 광 음향 프로브와 관심 영역의 동적 생물학적 이벤트를 시각화하는 실험 동안 동일한 위치에 촬상 부를 유지한다. </li><li> 관심 영역에서의 동적 분배를 추적하기 위해 조영제를 주입한다. NOTE : 마우스 우리의 실험에서는, 인도 사이 아닌 그린 (ICG)는 콘트라스트 개선을 위해 사용되었다. 일반적인 지침, 10 nmol의 0.4 mg의 양으로서 / kg의 ICG는 생체 내에서 멀티 스펙트럼 optoacoustics 검출 가능한 콘트라스트를 생성하기 위하여 마우스 순환 내로 도입되어야한다. 참고 : 조영제가 있어야합니다각각의 기관에 의한 인간 및 / 또는 동물의 사용이 승인. </li></ol> 5. 실험을 마무리 <ol><li> 레이저를 중지합니다. </li><li> 몇 군데 지역에서 광 음향 프로브를 제거합니다. 동물 실험의 경우, 마취 공급을 중지합니다. </li><li> 따뜻한을 유지하고 완전히 마취에서 회복 될 때까지 다른 동물과의 접촉을 방지하기 위해 적외선 히터에서 동물을 배치합니다. 마취에서 회복하는 동안 무인 동물을 방치하지 마십시오. </li></ol> 6. 오프라인 데이터 처리 <ol><li> 데이터 처리에 사용되는 소프트웨어 애플리케이션에서 취득한 광 음향 신호를 포함하는 파일 (들)을로드. </li><li> 각 프레임과 각 파장에 대한 광 흡수의 체적 화상에 대응하는 입체 매트릭스 어레이를 얻기 위해 재구성 알고리즘을 사용한다. 참고 :이 바람직하다 재건의 경우는 heterogene 등의 왜곡 요인에 대한 회계 알고리즘을 사용하는묘화 객체 시달 및 감쇠는, 순서대로 최종 대역폭 및 검출 소자 및 광 조사량 변동의 기하학적 형상의 영향은 흡수 된 에너지의 분포의 양적인 표현을 얻었다. </li><li> 각각의 다중 파장 프레임, 샘플에 존재하는 각 흡수 물질에 대한 광 흡수를 나타내는 입체 매트릭스 어레이들의 새로운 집합에서 수득 unmixing 알고리즘을 사용한다. </li><li> 필요한 경우, 추가의 생물학적 관련 파라미터의 시각화 및 판독을 용이하게하기 위해 광 흡수 분포를 나타내는 행렬 어레이를 처리한다. </li></ol>

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The authors have nothing to disclose.

작은 동물 연구에서 광 음향 영상 기술이 제공하는 독특한 장점은 예를 들어 구상 진단 및 치료 모니터링 응용 프로그램의 번호와, 임상 적으로 기술 번역에 대한 강한 의욕을 만들었습니다., 유방암, 피부 암, 염증이나 말초 혈관 질환. 그러나, 효과적인 단층 화상 취득 형상을 작성하는 조명 소스 및 검출 소자의 충분한 수에 의해 포위 될 수있는 마우스 또는 작은 동물, 대조적으로, 인체 ?...

기술 1-5에서 제공하는 독특한 장점을 악용 새로운 애플리케이션의 다양성을 포괄하는 출판물의 적 증가에 의해 나타난 바와 같이 광 음향 (음향) 영상은, 생물학 및 의료 연구 지역 사회에서 관심을 성장 끈다. 특히, 지금까지 빛의 확산 제한을 초과하는 깊이에서 높은 시공간 해상도의 이미지 스펙트럼 독특한 사진 흡수 에이전트 용량은 기능 및 분자 영상 6-10에 대한 전례없는 기능을 엽니 다. 실제로 임상에 광 음향 기술의 번역은 진단 및 많은 질병의 치료 모니터링에 유망한와 함께 제공됩니다. 그러나, 광학적으로 산란 광자 흡수 조직 및 광 음향 현상과 관련된 일반적 약한 응답의 제한된 전달은 방법의 적용 깊이를 제한한다. 그 결과, 휴대용 optoa내시경 시스템은 천연 오리피스 (13)를 통해 그것들을 삽입하여 본체 내에서 이미지를 제공하기 위해 사용되는 동안 coustic 프로브 본체 (11, 12)의 외부에서 접근 화상 부분이 시도되어왔다. 이러한 여성의 유방과 같은 인체의 일부 낮은 흡수 부분은, 또한 단층 광 음향 스캐너 (14, 15)에 의해 액세스 할 수 있습니다. 그것은 마찬가지로 초음파에 큰 융통성을 가능로서 특히 관심 휴대용 접근법이다. 여기서, 광 음향 영상에 대한 일반적인 초음파 선형 배열 프로브의 적응은 주로 초음파와 optoacoustics 사이의 단층 영상 요구 사항에 근본적인 차이, 도전 남아있다. 표준 초음파 높은 프레임 레이트가 순차적으로 활성화되지만 kHz의 범위에서 높은 펄스 반복 주파수를 이용하는 방식을 송수신 실시간 삼차원 광 음향 이미징 단일의 int로부터 체적 단층 촬영 데이터의 동시 수집함으로써 달성된다레이저 펄스를 errogating. 따라서, 고품질 광 음향 영상은 이미지화 된 물체에 가장 큰 가능한 고체 각도로부터 3 차원 데이터의 획득을 의미한다. 최근에, 우리는 실시간으로 16에서 3 차원 (부피) 영상에 대한 최초의 휴대용 광 음향 프로브를 소개했다. 시스템은 90 °의 각도를 덮는 구면 (도 1A에서 청색 도트)에 배치 된 압전 소자 (256)의 2 차원 어레이를 기반으로한다. 대략 3 × 3mm (2)의 개별 요소들의 크기뿐만 아니라 방향과 주파수 대역폭 (약 2-6 메가 헤르츠)도에 구의 중심 (검은 큐브 주변 센티미터 규모 볼륨에서 유효 신호 수집을 보장 1A). 촬상 영역의 여기 광은, 어레이의 중앙 원통형 공동에 삽입 관통 섬유 다발이 제공되도록되어 어떤 파장 susc섬유 다발을 통해 전송되는 eptible 이미징에 사용될 수있다. 광섬유 다발과 함께 트랜스 듀서 어레이의 실제 사진은도 1b에 도시된다. 레이저의 펄스 반복 주파수에 의해 결정된 프레임 율 실시간 이미징 상기 그래픽이 활성화 될 수 있도록 효율적으로 여기 및 신호를 동시에 검출은, 단발 여기 (하나의 레이저 펄스)와 깊은 조직 이미징을 허용 프로세싱 유닛 (GPU) 재건 절차 (17)의 구현입니다. 투명 폴리에틸렌 막 (도 1c)와 원통형 케이싱은 음향 투과 액 매체 (물)를 둘러싸도록 트랜스 듀서 어레이에 부착된다. 멤브레인은 또한 탄성 겔 의해 조직에 결합된다. 휴대용 동작 모드에서 사용되는 것과 프로브의 광 음향 영상은도 1d에 도시되어있다. 입증 THR임상 진단 및 잠재적 인 응용 프로그램의 수에 대한 중요한 장점과 함께 제공되는 실시간 기능 이미징 능력과 결합 EE 차원 휴대용 광 음향 이미징, 말초 혈관 질환 등의 다양한 표시, 림프계 장애, 유방암, 피부 병변에 대한 구상하고 있습니다 염증이나 관절염 (18). 더욱이, 고속 촬상 용량은 고정 된 위치에 배치 된 프로브와 동적 생물학적 이벤트의 시각화를 가능하게한다. 빠른 파장 동조 광 파라 메트릭 발진 (OPO) 레이저 기술과 결합하여,이 방식은 광 흡수제의 생체 분포를 실시간 이미징을 허용한다. 이에 따라, 새로운 가능성이 동등하게 예를 들면, 작은 동물 이미징 애플리케이션에 등장 할 수있다. 조직의 혈류 역학 연구에서, 생체 내 세포 추적, 약물 동력학, 장기 관류의 시각화, 종양 및 심장 혈관 시스템, 또는 신경 영상의 분자 영상을 대상으로.

<table border="0" cellpadding="0" cellspacing="0" width="1663">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Name of Material/ Equipment</td>
			<td style="width:389px;">Company</td>
			<td style="width:171px;">Catalog Number</td>
			<td style="width:777px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Optical parametric oscillator (OPO)-based laser</td>
			<td style="width:389px;">Innolas Laser GmbH, Krailling (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The laser provides laser pulses with a duration around 10ns and an energy up to 80mJ. 
			The wavelength is tunable between 680-950nm.</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:327px;">Spherical array of piezocomposite detectors</td>
			<td style="width:389px;">Imasonic SaS, Voray (France)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The array consists of 256 piezoelectric sensors distributed on a spherical surface. 
			Each element has dimensions 3x3mm2, a central frequency of 4MHz and a bandwidth of 100%.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data acquisition system (DAQ)</td>
			<td style="width:389px;">Falkenstein Mikrosysteme GmbH, Taufkirchen (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The DAQ simultaneously acquires 256 signals at 40 megasamples per second and 2030 samples. 
			The input impedance is 1MW.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Fiber bundle</td>
			<td style="width:389px;">CeramOptec GmbH, Bonn (Germany)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The bundle consists of 480 individual fibers randomly distributed in the input and output. 
			The numerical aperture of each individual fiber is 0.22.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Athymic Nude mouse</td>
			<td style="width:389px;">Harlan Laboratories (The Netherlands)</td>
			<td style="width:171px;">Athymic nude - Foxn1nu</td>
			<td style="width:777px;">The mouse was 8 weeks old (adult) at the time of the experiment. 
			The ethical protocol was approved by the Bavarian goverment (number 55.2.1.54-2632-102-11)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Bepanthen cream</td>
			<td style="width:389px;">Bayer AG (Germany)</td>
			<td style="width:171px;">N/A</td>
			<td>Vet ointment to protect the eyes during anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Data processing software</td>
			<td style="width:389px;">Matlab (Mathworks, Natick, MA, USA)</td>
			<td style="width:171px;">custom-made</td>
			<td style="width:777px;">The data processing software was devoped at our institute. 
			It allows reconstruction at each wavelength and multi-wavelength unmixing, as well as further data processing.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:327px;">Water-enclosing part</td>
			<td style="width:389px;">N/A</td>
			<td style="width:171px;">custom-made</td>
			<td>This part contains the water that acts as an acoustic coupling medium between skin and transducer elements</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Indocyanine green (ICG)</td>
			<td style="width:389px;">PULSION Medical Systems SE</td>
			<td style="width:171px;">N/A</td>
			<td style="width:777px;">ICG-PULSION (active ingredient: indocyanine green dye) is a drug used in cardiac, circulatory and micro-circulatory diagnostics, liver function diagnostics and ophthalmic angiography diagnostics.</td>
		</tr>
	</tbody>
</table>

universal hand-held three-dimensional optoacoustic imaging probe for deep tissue human angiography and functional preclinical studies in real time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

설명 체적 휴대용 광 음향 프로브의 기능을 보여주는 대표적인 결과는,이 섹션에 전시된다. 모든 경우에, 피부 표면에 광 조사량 20 mJ을 / cm 2 (19)의 안전 노출 한계 이하로 유지 하였다. 주변 인간 혈관 추적 실시간 프로브의 성능을도 2에 전시된다.이 실험의 과정에서, 프로브는 서서히 레이저 동작 800 nm의 단파장 건강한 인간 지원자의 손을 따라 스캔 한...

Watch this Scientific Journal Video about Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time at JoVE.com

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

범용 실시간으로 깊은 조직 인간의 혈관 조영술과 기능 전임상 연구를위한 3 차원 광 음향 이미징 프로브를 손 개최

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

universal-hand-held-three-dimensional-optoacoustic-imaging-probe-for

Research

JoVE Journal

Bioengineering

10.6K 조회수.  Helmholtz Zentrum München. We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

비디오: Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

입체 광학 해상도 Photoacoustic 현미경

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

연구

생체공학

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

전도성 잉크의 평면 및 3 차원 인쇄

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

생리 흐름 조건에서 순환 세포의 케모카인 - 감독 넘쳐을 연구하는 소설 입체 흐름 챔버 장치

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

입체 잉크젯 인쇄 기술을 사용하여 인간의 연골 조직 제작

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

인간의 장 점막의 다세포 3 차원의 Organotypic 모델의 개발은 미세 중력 하에서 성장

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

3 차원 조직 설계 정렬 사이토 네트워크 발달 메커니즘을 정리 하 고 신경 재생을 촉진 하

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

조직 공학 응용 프로그램에 대 한 제어 형태학과 3 차원 Poly(ε-caprolactone) 장비의 전기 글 녹아

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Hand-held Clinical Photoacoustic Imaging System for Real-time Non-invasive Small Animal Imaging

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, we report a combined photoacoustic and clinical ultrasound imaging system by integrating an ultrasound probe with light delivery for small animal imaging. We demonstrate this by showing sentinel lymph node imaging in small animals along with minimally invasive real-time needle guidance. A clinical ultrasound platform with access to raw channel data allows the integration of photoacoustic imaging leading to a handheld real-time clinical photoacoustic imaging system. Methylene blue was used for sentinel lymph node imaging at 675 nm wavelength. Additionally, needle guidance with dual modal ultrasound and photoacoustic imaging was shown using the imaging system. Depth imaging of up to 1.5 cm was demonstrated with a 10 Hz laser at a photoacoustic imaging frame rate of 5 frames per second.

A clinical handheld photoacoustic imaging system will be demonstrated for real-time non-invasive small animal imaging.

휴대용 임상 Photoacoustic 실시간 비-침략 적 작은 동물 이미징을 위한 이미징 시스템

Translation of photoacoustic imaging into the clinic is a major challenge. Handheld real-time clinical photoacoustic imaging systems are very rare. Here, ...

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 ...