The authors acknowledge support from a Translational Breast Cancer Research Grant (14-60-26-BROC to AB) from the Breast Cancer Research Foundation and the American Association for Cancer Research.

동물의 주제를 사용하는 절차는 텍사스 오스틴 대학의 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1. 수술 전 준비 <ol><li> 각각의 토끼의 체중을 기록한다. 모든 임상 시험과 같이, 잠재적 인 독성을 평가하기 위해 정기적으로 동물의 무게를 모니터링한다. </li><li> 전에 토끼를 마취로, 시중에서 판매하는 초음파 젤 (30)의 500 XG에 스핀 50 ML 원뿔 관을 채우기; 원심 분리 완료시 겔에 가시적 인 거품이 없어야합니다. </li><li> 0.75 ㎎ / ㎏의 투여 량으로 근육 내 아세 프로 마진, 0.1 ㎎ / ㎏의 용량으로 피하 글리코을 관리하고. 생체 신호와 동작을 모니터링하면서 적용하려면 진정을 위해 15 ~ 20 분을 기다립니다. 참고 : 글리코은 서맥을 방지하고 호흡과 GI 분비물을 감소시키는 항 콜린성 에이전트입니다. 아세 프로 마진은 premedicati 역할을하는 진정제입니다마취에. </li><li> 케타민 35 ㎎ / ㎏과 kg의 자일 라진 피하 마취로 5 mg을 /를 관리 할 수 ​​있습니다. 그러나 운전자가 경험하게되고 케타민 3 밀리그램의 / kg 15 mg의 약물 투여 량을 감소, 더 빠르게 췌관 전달을 수행 할 수 / 자일 라진 피하의 kg; 이것은 마취 시간 및 마취에서 회복하는 동물에 필요한 시간을 단축한다. </li><li> 확인하고 심장 박동, SPO 2, 온도, 호흡, 및 점액 막 색 매 15 분을 기록. 두 눈에 눈 윤활제를 적용합니다. 참고 : 적절한 훈련을받은 및 관리 또는 마취를 모니터링해야 그 기관의 IACUC에 의해 승인 된 인원 만. 수의사 마취 모니터의 사용은 생체 신호를 수집하고 권장에 도움이 될 수 있습니다. 수의학 마취 모니터 그러나, 수동으로 동물에게 15 분마다 체크를 할 필요를 대체하지 않는다. f를 제조업체의 지침을 참조하십시오또는 수의학 모니터의 적절한 사용. </li><li> 부드러운 발가락 핀치에 의해 마취의 발병을 확인; 토끼는 계속하기 전에 비 응답해야합니다. </li><li> 조심 사타구니 유두의 제 3의 형태 및 제 4 쌍의 주변에 토끼의 꼬리 복부를 면도. </li><li> 제거 된 머리의 대부분으로, 면도 영역에 오버 - 더 - 카운터 제모 크림을 적용합니다. 따뜻한 물에 젖은 축축한 종이 타월을 사용하는 응용 프로그램 후 크림 10 분을 제거합니다. 참고 : 토끼 피부가 민감한 / 매우 깨지기입니다. 제모 크림은 더 이상 10 분 동안 사용되어야한다. 실제로, 5 분으로 &quot;테스트 지점&quot;을 시도하고 테스트 스폿이 기간이 필요한 더 긴 시간을 나타내는 경우에만 이상에두고 안전하다. </li><li> 주사 부위를 청소하기 위해 알코올을 적신 거즈 패드 영역을 닦습니다. </li><li> 순환되는 온수 담요와 흡수 패드 늘어서 V 자형 골에서의 배부에 토끼를 놓습니다. </li></ol> 2. 홍보조영제의 eparation <ol><li> 제조업체의 지시에 따라 타겟팅되지 않은 대조 시약을 재구성한다. 최대 피펫 아래로 부드럽게 혼합. 주 :이 프로토콜에서 사용되는 조영제 4 실온에서 안정 - 재구성 후 6 시간. 조심스럽게 각 검색 사이의 유리 병 바위. 주 : 덕트의 수에 의존 할 필요 용액의 부피가 주입된다. 솔루션은 성인 뉴질랜드 화이트 토끼 (Oryctolaguscuniculus) 중 하나 도관 트리를 채우기에 충분하다의 토끼는 유두 당 4 유관 구멍, 0.2 mL로있다. 이에 따라, 0.8 mL의 총 부피는 하나의 유선으로 전달 될 수있다. </li></ol> 3. 췌관 배달 <ol><li> 주입 할 수있는 적절한 유두를 찾습니다; 동물이 그것의 배부에 위치 할 때 쉽게 가시화 될 때 사타구니 유두의 3 번째 및 4 번째 쌍은, 사용하는 것이 좋습니다. </li><li> 로 멸균 0.9 % 식염수를 0.2 mL의 부하22 G 바늘 1 mL의 루어 잠금 투베르쿨린 주사기. 제대로 식염수를 주사기에 한 번 22 G 바늘 폐기 및 멸균 25 게이지 바늘로 교체합니다. 부드럽게 거즈 패드에 85 % 이소 프로필 알코올로 영역을 닦습니다. </li><li> 바늘 위로 동물의 몸에 주사기 병렬의 경사에 의해, 유두의 측면에 바늘의 베벨이 삽입 천천히 식염수 0.1-0.2 mL의 주입; 이것은 유관 개구의 더 나은 시각화 수 있습니다. </li><li> 로드 1 mL의 루어 잠금 투베르쿨린 주사기로 주입 용액 0.2 mL를. </li><li> 엄지와 검지 손가락으로 부드럽게 유두를 잡고 관내 주입을 위해 위치를 약간 들어 올려; 착용 안구 루페는 도관 개구부를 시각화에 도움이된다. </li><li> 유두의 상승 위치를 유지하면서 조심스럽게 25 G 뭉툭한 팁 바늘을 사용하여 관심있는 덕트를 cannulate. </li><li> 삽관 후, 부드럽게 무딘-t의 중심에있는 루어 락 주사기를 비틀제자리에 고정 될 때까지 IP를 주입 바늘. </li><li> 유두를 들어 올리고 빠르게 덕트 내에서의 유체 이동에 의한 손상을 최소화하기 위해 천천히 용액을 주입; 용액을 주입 할 때 짧은 시간에 저항이 있어야합니다. </li></ol> 4. 초음파 이미징 <ol><li> 관심 영역의 피부에 원심 분리 초음파 젤의 자유로운 금액을 적용합니다. 이러한 이미지 품질을 손상되므로, 겔에 기포가 없도록. </li><li> 6mm에 이미징 깊이를 설정합니다. 겔과 접촉하는 21 MHz의 진동자를 배치하고 B 모드에서 관심 영역을 스캔. 도관 개구 덕트에 걸쳐 포함하는 검사 영역의 조영제를 관찰한다. 주의 : 이러한 설정은 특정 음향 초음파 장치와 함께 사용하기 위해 개발되었다. 자세한 내용은 재료의 표를 참조하십시오. 이는 유방을 최적화하기 위해 송신 전력 및 다른 이미징 시스템의 촬상 깊이를 조정하는 것이 필요할 수있다그랜드 시각화. </li><li> 거즈 패드 동물의 피부에서 초음파 젤을 제거합니다. </li></ol> 5. 수술 후 케어 <ol><li> 유두 영역이나 주변 조직에 외상 흔적이 없어야하고, 유두 주변 영역에서 팽창하는 것으로 유방 지방 패드에 주입보다는 성공적인 췌관 주입 나타냄 주입 부위를 관찰한다. </li><li> 흉골 위치에 토끼를 놓습니다. 필요한 경우, 정맥 내 귀 가장자리 정맥 요힘빈 0.2 ㎎ / ㎏을 제공; 이 자일 라진의 효과를 반전하고, 동물이 마취로부터 더 빨리 복구하는 것을 허용 할 것이다. </li><li> 동물이 응답하는 경보이며, 흉골 위치에 유지 될 때까지 토끼에게 회복 기간 동안 15 분마다 모니터. 참고 :이 절차는 조직 손상이나 붓기가 발생하지 않아야합니다. 적색 또는 관찰 붓기 경우 동물이 경고와 수되면, 멜 록시 캄 0.1 ~ 0.2 ㎎ / ㎏ PO의 복용량을 관리구두 약을 복용합니다. 추가 지침에 대한 기관의 수의학 직원에게 문의하십시오. </li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Addressing+Overdiagnosis+and+Overtreatment+in+Cancer:+A+Prescription+For+Change.">Addressing Overdiagnosis and Overtreatment in Cancer: A Prescription For Change.</a> Lancet Oncol. 15 (6), e234-e242 (2014).</li></ol>

The authors have nothing to disclose.

토끼 유방 선으로 관내 전달하는이 방법은 중요한 염료 및 치료를 포함하여, 초음파 대조 시약 및 기타 수용액에 사용될 수있다. 이전의 연구는 호르몬 17, 18, 19의 췌관 배달을 증명하고있다. 설치류 모델 핵산 (8)의 관내 전달, 화학 요법 제 6, 7, 나노 캐리어 <sup clas...

<html> 치료제의 관내 전달은 설치류 모델에서 초기 및 인간 실험 3, 4, 5, 6, 11, 12에서 검토되고있다. 공부의 최근 단계는 췌관 카보 플 라틴 또는 침습성 암 (2)의 치료를 위해 유방 절제술을 기다리고 여성의 췌관 페 길화 된 리포좀 독소루비신의 안전과 가능성을 보여 주었다. 관내 전달을 위해 이전 프로토콜은 마우스와 랫트 젖샘 6, 7, 8, 9에 대해 개발되어왔다. 연구 목적, 관내 종양 세포를 주사하고 암 유전자의 렌티 바이러스 벡터 전달에도 설치류 모델에서 수행 된REF &quot;&gt; 13, 14, 15, 16. 그러나 관내 전달 프로세스의 생체 내 모델에서 이상적 치료 화합물의 신규 클래스의 개발을 허용하고 임상 평가를 용이하게한다. 설치류와 인간 사이의 해부학 적 차이는 이들의 번역을 복잡했다 연구. 마우스와는 달리, 각각의 덕트는 별도의 젖꼭지에서 종료하는, 인간의 가슴은 5 ~ 9 독립적 인 유관 시스템, 유두에서 끝나는 별도의 개방 각으로 구성되어 있습니다. 토끼 유방 땀샘은 네 개의 독립 유관 시스템, 하나의 젖꼭지에 네 개의 구멍 중 하나를 통해 각각 개별적으로 접근 항구. 토끼 모델은 더 밀접하게 일치 인체 해부학 및 더 중요한 상황에서 관내 약물 전달 연구를 허용한다. 여기, 우리는 관내 배달을 평가하기 위해이 기술을 사용합니다. (A)의 공동 투여중요한 색소는 피부로 시각화를 허용하고있어서 간단하고 신속하게 확인을 제공한다. 일부 애플리케이션의 경우, 덕트 고해상도 매핑은 바람직 할 수있다. 우리는 비 대상 대조 시약 관내 전달을 통해 여기 덕트의 초음파 영상을위한 프로토콜을 제시한다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MicroMarker non-targeted contrast reagent</td>
			<td>VisualSonics</td>
			<td>VS-11694</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer Lock 1mL Syringes</td>
			<td>BD</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycopyrrolate 0.2mg/mL</td>
			<td>Wedgewood Compounding Pharmacy</td>
			<td>GLYCOP-INJ013VC</td>
			<td>6 month shelf life, supply may be limited.&#160;</td>
		</tr>
		<tr>
			<td>Atropine Sulfate 0.5 mg/mL</td>
			<td>Animal Health International</td>
			<td>15320764</td>
			<td>If glycopyrrolate is unavailable. Not to be combined with glycopyrrolate.</td>
		</tr>
		<tr>
			<td>Ketamine HCL 100mg/mL</td>
			<td>Animal Health International</td>
			<td>21250699</td>
			<td><a href="http://www.animalhealthinternational.com/">http://www.animalhealthinternational.com/</a></td>
		</tr>
		<tr>
			<td>Acepromazine 10mg/mL</td>
			<td>Animal Health International</td>
			<td>17640541</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 20mg/mL</td>
			<td>Animal Health International</td>
			<td>20101547</td>
			<td></td>
		</tr>
		<tr>
			<td>Yohimbine 0.2mg/mL</td>
			<td>Animal Health International</td>
			<td>14588965</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Removing Cream</td>
			<td>Veet</td>
			<td></td>
			<td>Sensitive skin solution. Available through local retailers.</td>
		</tr>
		<tr>
			<td>Blunt tip infusion needles</td>
			<td>Sai Infusion Technology</td>
			<td>B14-50</td>
			<td><a href="http://www.sai-infusion.com/collections/blunt-needles">http://www.sai-infusion.com/collections/blunt-needles</a></td>
		</tr>
		<tr>
			<td>Veterinary Pulse Oximeter</td>
			<td>EdanUSA</td>
			<td>VE-H100B</td>
			<td><a href="http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html">http://www.edanusa.com/Product/VE-H100B-Veterinary-Pulse-Oximeter.html</a></td>
		</tr>
		<tr>
			<td>Warm Water Pump</td>
			<td>Gaymar</td>
			<td>TP700</td>
			<td></td>
		</tr>
		<tr>
			<td>Warm Water Blanket</td>
			<td>Animal Health International</td>
			<td>21232696</td>
			<td>Maxi-Therm Lite Warming Pads</td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>VisualSonics</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
	</tbody>
</table>

intraductal delivery to the rabbit mammary gland

유방암 췌관 국소 치료는 종양에 대한 효율적인 전달 및 감소 된 독성 및 부작용이 1, 2, 3, 4, 5, 6, 7을 포함한 잠재적 인 이점을 제공한다. 이러한 치료가 더 널리 적용 할 수 있습니다 전에 그러나, 몇 가지 과제가 남아있다. 적절한 동물 모델의 개발과 췌관 치료제의 유효성 검사는 환자 췌관 치료 전략의 개발을 용이하게한다. 마우스 유방 선 널리 유선 종양의 발달 및 모델 시스템으로 사용되었지만, 해부학 인간 동맥 구별된다. 더 큰 동물 모델은 같은 토끼로, 유선 구조 및 췌관 치료 개발을위한 더 나은 모델이 될 수 있습니다. C에서마우스에 ontrast,하는 열 유관 나무 공간적으로 분리 된 유두의 각 종단은 토끼 유방 동맥은 더 밀접하게 한 젖꼭지에 별도의 구멍을 통해 그 출구 여러 중복 유관 시스템과, 인간의 동맥과 유사한 신체의 축을 따라 배포됩니다. 여기서는 직접 유선 토끼 덕트로 고해상도 초음파 영상과 함께 전달 자체의 시각화를위한 시약의 전달을위한 최소 침습적 방법을 제시한다.

여기서는 토끼 유방 덕트 대조적 시약의 관내 전달은 조직 (도 2)에 충격없이 달성 될 수 있다는 것을 보여준다. 토끼에서, 네 개의 별도의 유관 시스템은 하나의 젖꼭지에 수렴 따라서 액세스 할 수 있으며,이 방법을 사용하여 개별적으로 몇 군데. 개인 유관 구멍이 쉽게 나타나는; 도 2b에서 유관 덕트에 인접한 제 2 도관 개구를 표시 화살촉을 확?...

Watch this Scientific Journal Video about Intraductal Delivery to the Rabbit Mammary Gland at JoVE.com

Intraductal Delivery to the Rabbit Mammary Gland

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

토끼 유선으로 관내 배달

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

intraductal-delivery-to-the-rabbit-mammary-gland

Research

JoVE Journal

Bioengineering

9.2K 조회수.  The University of Texas at Austin. Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents. 

비디오: Intraductal Delivery to the Rabbit Mammary Gland

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

Langendorff - perfused 토끼 하트의 Multiparametric 광 매핑

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

연구

생체공학

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

의 순환 압력 생물 반응기 설계 예 VIVO</em대동맥 심장 밸브> 학습

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

줄기 세포 - 로드된 키토산 Microspheres의 배달을 위해 콜라겐 히드로겔을 구축

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

마이크로 소용돌이를 이용한 순차 분자 전달을위한 Electroporator을

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

전기 방사를 적응하는 것은 트라이 계층에 대한 세 가지 독특한 환경을 제공하기 위해<em&gt; 체외</em&gt;기도 벽의 모델

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

인간의 유선 상피의 호르몬 응답 3D 문화 모델

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

양이온과 음이온 리포좀을 사용 CNS에 치료의 siRNA 배달

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

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

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

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

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

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

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Retroductal 나노 Murine Submandibular 동맥에 주입

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

유 방 땀 샘에서 조사 된 유기 체의 성장과 특성화

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...