저자는 토스카나 지역 (POR CREO FESR 2007-2013, 반도 유니코 R &amp; 2012 S)에 의해 투자 FORTE 프로젝트, 부분적으로 연구 활동을 지원하는 유럽 연합 (EU) FP7 ECHORD ++ 실험 LA-장미, 그리고 부여 된 FP7 BiophotonicPlus 프로젝트 &quot;라이트&quot;감사드립니다 토스카 지역별.

이 연구는 병원 윤리위원회의 전향 적으로 승인 하였다; 동의서를 얻었다. 이 연구는 헬싱키 선언의 원칙을 준수했다. 1. 기증자 내피 준비 <ol><li> 실온에서 지역 안구 은행에 의해 제조 된 기증자의 각막을 사용합니다. </li><li> 수술실에서의 배달 컨테이너에서 기증자의 각막을 당겨 각막 전송에 사용되는 조직의 보존과 영양에 대한 솔루션을 구별. </li><li> 액체가 흘러 변수 압력을 만들 수있는에 인공 전방, 이상 기증자 corneo - 공막 림을 놓고; 조직 유지 헤드와 각막을 커버합니다. </li><li> 각막 보존 및 영양 액체로 가득 주사기에 3 방향 커넥터를 통해 인공 전방 (AAC)를 연결합니다. </li><li> 장소에 안전하게 고정을 유지하기 위해 압축 링을 사용합니다. </li><li> SYR를 사용하여최적 압력 (1.7 참조​​)에 도달 할 때까지 조직 보존 용액으로 전방을 채우 인게은 전방 내 압력을 유지하기에 접속 AAC. 커넥터를 닫습니다. </li><li> 손가락으로 전방 내부의 압력을 테스트합니다. (범위 60 ~ 70 mmHg로에서) 오른쪽 내부 압력에 도달 할 때까지 내부 압력을 변경합니다. 3 방향 커넥터습니다. </li><li> 빛 간섭 Thomography OCT ()와 도너 각막의 두께를 측정한다. 악기 광학 앞에 고정 된 위치에 그 각막과 전방을 유지하고 전체 두께 10월 수집을. </li></ol> 기부자 내피 2. 펨토초 레이저 준비 <ol><li> 펨토초 레이저 장치에서 기증자의 각막의 압평을 수행합니다. 후방 사이드 컷, 전체 층상 및 전방 사이드 컷 : 세 이후의 상처와 기증자의 조직을 잘라 femtosec 레이저를 사용합니다. 세 인하에 대해 다음 매개 변수를 설정합니다.<ol><li> 전체 라멜라 컷 (래스터 패턴, 아웃 시작)의 경우, 항상 95 μm의 감산 (1.8에 기재된 바와 같이 OCT를 사용하여 측정) 공여자 각막의 얇은 포인트에 대응하는 절단 깊이를 설정한다. 범위 (작업 깊이에 따라 다름) 0.8-0.9 μJ의 펄스 에너지를 설정합니다. 8.7 mm의 직경을 설정합니다. 2 μm의 접선 장소의 분리를 설정합니다. 2 μm의 반경 자리 분리를 설정합니다. </li><li> 전방 사이드 컷의 경우, 이전의 전체 라멜라 컷 30 μm의 깊은 후방 깊이를 설정합니다. 2.10 μJ에 펄스 에너지를 설정; 8.6 mm의 직경; 층 분리 동안 3 μm의 3 μm의 자리 분리. </li><li> 후방 사이드 컷의 경우, 후방 측의 전방 깊이가 전체 라멜라 컷보다 전방으로 30 μm의 컷 찾습니다. 900 μm의 후방 깊이를 설정합니다. 2.10 μJ에 펄스 에너지를 설정; 8.3 mm의 직경; 2 μm의 자리 분리; 2 μm의 층 분리. </li></ol></lI&gt; </ol> 3.받는 사람 눈 준비 <ol><li> 수술 환자를 준비합니다. 블레이드를 사전 교정 4.0 mm의 12시 한 4.00 mm의 각막 절개를합니다. 2시에 배치 1.2 mm의 사전 교정 블레이드 하나 윤부 천자를 확인합니다. 30 ° 자상 칼과 6시 방향에서 다른 윤부 천자를 확인합니다. </li><li> 2시 천자를 통해, 환자의 전방에 전방 메인테이너를 삽입합니다. </li><li> 전용 후크 한 8.2 mm 직경의 원형 descemetorexis을 수행합니다. </li><li> 후방 기질에서 데스 메 막을와 내피를 벗겨과 데스 메 후크와 조직을 제거합니다. </li></ol> 4. 발색단 준비 <ol><li> 1.5 ml의 microcentrifuge 관에서 인도 사이 아닌 그린 분말 1 mg을 넣어. </li><li> 멸균 9 mg을 추가 (ICG 수용액은 W / W 10 %). </li><li> 수동 금속 교반기 ICG 가루와 물을 혼합한다. </lI&gt; </ol> 기부자 내피 5. 염색 <ol><li> 분사 장치 표면과 접촉하는 내부면이 되네요 인젝터의 넓은 부분에 도너 내피을 넣어. </li><li> 주걱을 사용하여, 그 주변 부분에, 발색 용액 도너 내피의 내측 얼룩. 제대로 스테인드 조직은 균일 한 녹색 색상을 가지고있다. 단계 5.3을 시작하기 전에 3 분을 기다립니다. </li><li> 되네요 - 인젝터의 앞쪽 부분에 기증자의 내피 세포를 당겨, 압연. </li></ol> 6. 기증자 내피 삽입 <ol><li> 잡고 내피 lenticule에 대한 외상성 동축 집게를 사용하여 접힌 기증자의 내피 세포를 삽입합니다. </li><li> 전방 메인테이너를 제거합니다. </li><li> 각막 절개와 나일론 10.0 단일 스티치와 천자 (3.1 단계에서 수행)를 봉합. </li><li> 공여 조직을 전개하고 수신자 각막에 대해 그것을 누르는 기포 주입한다. 공기 방울 미리보기 다운로드합니다letely 전방 공간을 채우기. </li></ol> 7. 레이저 용접 <ol><li> 후크 또는 주걱으로 각막의 외부로부터 공기 기포 이동 내부로부터, 수신자 각막의 중앙에 도너 lenticule 배치. </li><li> 개구 수 (NA) 0.22과 같이 300 μm의 코어 직경 멸균 광섬유 갖춘 810 nm에서 발광 다이오드 레이저를 사용한다. </li><li> 안구 외측 섬유 팁을 유지하고, 투명한 각막 조직을 통해 염색 내피 향해 레이저 광을 전달한다. 광섬유 팁은 비접촉 구성이다. </li><li> 단일 지점 방출 모드, 70 ~ 80 밀리 초 펄스 폭, 펄스 당 35 ~ 40 엠제이 : 레이저에 대해 다음 설정을 사용합니다. 그는 느가에 빔을 목표로합니다. </li><li> 기증자 내피 세포의 염색 주변에 하나의 레이저 스팟을 제공합니다. 순차적으로 스폿 배달 : 최종 형태는 도너 lenticule의 주위에 스폿의 링이다. 두 adja 사이의 거리센트 명소 센터 스폿 직경의 두 배입니다. </li><li> 토 브라 마이신 0.3 % 및 0.1 % 덱사메타손 안과 현탁액과 함께, 환자의 눈에 콘택트 렌즈를 적용. </li></ol>

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The authors declare that they have no competing financial interests.

&quot;모든 레이저&quot;내피 이식은 원래의 접근 최소 침습 각막 이식하는 것입니다. 프로토콜 내에서 설명하는 모든 절차는 멸균 장갑, 가운, 마스크와 모자의 사용으로 수술하는 동안 일반적인 관행이다 위생과 살균 과정을 관찰, 수술 실에서 실시 하였다. ICG 용액은 곧 도너 내피 염색에서의 도장 전에, 수술실에서 제조 하였다. ICG 분말, 물 및 염색 용액을 제조하는 데 사용?...

본 연구에서 우리는 공여 조직 및 수신자 베드 상을 용접 근적외선 레이저 다이오드를 제조하는 펨토초 레이저의 사용을 기반 각막 내피에 원래 방식을 제시한다. 공여자 각막의 수술 측정 올바르게 공여 조직 치수를 설계하는 것이 필요하다. 각막 내피는 내피 1,2- 질병 치료에 각막을 교체하는 최근에 제안되었다. 이 기술의 가장 큰 장점은 수술, 이식 거부의 감소 위험과 눈의 무결성을 보존하는 동안 각막 감소 마취를 관통에 대하여, 빠른 시력 회복이다. 주요 위험 인자는 수술 후 기증자 lenticule 탈구이다. 표준 기법이 기포 주입에 의해 유지되는 최종 위치에 도너 내피를 삽입함으로써 수행된다 : 어떤 봉합사 때문에, 생 물리적 및 기계적 치수 체인지의 사용되지내피 세포의 cteristics. 또한, 시력 회복으로 인해 이식 두꺼운 조직에 주로 있기 때문에 기증자와받는 사람의 조직 사이의 불일치 제한 할 수 있습니다. 여기에서 우리는 이러한 주요 문제를 극복 할 수있는 내피 각막 이식술을 수행의 절차를 제시한다. 도너 내피는 레이저 용접 기술을 사용함으로써 최종 위치에 확보 할 수있다. 이 제어 및 지역화 광열 과정이다 : 그것은 기증자 /받는 사람 인터페이스에서 유도 될 수있다. 그것은 지난 10 년 동안 공부하고 각막과 내피 3-5의 이식에 제안되었다. 근적외선 (파장 810 ㎚) 저전력 레이저 다이오드에 의해 방출은 상처 부위에서 생체 조직을 향해 전달된다. 각막이 파장에 자연적으로 투명하다 : 레이저 광을 흡수하여이 조직을 만들기 위해서는, 발색단 그것을 염색하는 것이 필요하다. 제안 된 염료이다 멸균 포화인도 사이 아닌 그린 (ICG)의 D 수용액. 우리는 각막 조직이 제대로이 ICG 제제로 염색 할 때, 810 nm의 6에서 흡수 피크를 나타내고 있음을 보여 주었다. 또한, ICG 널리 임상 진단에 사용되며 안전성은 이미 인간의 과목에서 입증되었다. 각막 염색은 다이오드 레이저 광 에너지를 흡수하여 생성 된 주요 효과는 용접 부위의 온도 상승을 제어한다. 아니 열 효과는 흠없는 조직에서 유도되지 않습니다. 온도의 향상은 냉각시 상처 벽 즉각적인 closuring와, 간질 콜라겐 가역적 열 변성을 유도한다. 이 레이저 용착 효과 첫째 백내장 및 각막 7,8 9,10 관통에서 입증되었다. 우리는이 논문에서 제시되는 최적화 방법은 각막 내피 세포의 응용 프로그램에 대한 연구되고있다. 제안 된 수술에서, 하나의 레이저 반점 (밀리의 지속 수십) ARE는 스폿 사이즈 (직경 ㎛의 수백) 내에 국부적 광열 효과를 초래 조직에 전달 : 유도 효과는 도너 / 호스트 인터페이스에 국한 콜라겐 광응고술 이루어진 하드 레이저 용접이다. 용접 부위 콜라겐 변성의 결과는 표준 기술에 따라서 (바늘)을 얻을 수 없다 봉합 효과를 제공하고, 도너와 호스트 조직 사이에 강한 접착력이다. 조직은 짧은 후속 (1 개월)에 그의 천연 거리는은 도너 및 / 숙주 조직과의 밀착성은 치료상의 초기 단계에 설치된 용접에 의해 향상된다. 두꺼운 공여 조직 이식 즉 내피 각막의 다른 주요 위험을 피하기 위해, intrasurgical 광 간섭 단층 OCT ()이 사용되어 정확한 절단 프로파일이 설계 될 수 있도록 상업 디바이스, 도너 각막의 두께를 측정femtosec 레이저. 제안 된 &quot;모든 레이저&quot;내피 이식 따라서이 최소 침습 수술의 임상 결과를 개선하는 것 같다.

<table><tbody><tr><td>Indocyanine Green</td><td>Pulsion Medical Systems, Germany</td><td>ICG-PULSION (http://www.pulsion.com/international-english/perfusion/icg-pulsion/)</td><td>Alternative product: IC-GREEN, Akorn&amp;nbsp; Inc., Lake Forest, Illinois- US (http://www.icginjection.com/)</td></tr><tr><td>Femtosecond Laser&amp;nbsp;</td><td>Abbott Medical Optics,&amp;nbsp; Abbott Laboratories Inc. Abbott Park, Illinois, USA</td><td>iFS150 (http://www.abbottmedicaloptics.com/products/refractive/ilasik/ifs-advanced-femtosecond-laser)</td><td></td></tr><tr><td>Optical Coherence Tomography (OCT)</td><td>Carl-Zeiss Meditec, Dublin, California- US (http://www.zeiss.com/meditec/en_de/home.html)</td><td>Visante</td><td></td></tr><tr><td>Diode Laser</td><td>E.l.En. Group s.pa., Calenzano-FI, Italy (http://www.elengroup.com/en/divisions/medical)</td><td>Mod. WELD 800</td><td></td></tr><tr><td>Artificial Anterior Chamber</td><td>CORONET, corneal graft products. Network Medical Products Ltd. Coronet House, Kearsley Road, Ripon, North Yorkshire, HG4 2SG, UK</td><td>Artificial Anterior Chamber (A.A.C.) with large and small tissue-retaining heads. Code 51-935 (http://www.networkmedical.co.uk/ophthalmic_artificial_&lt;br /&gt;anterior_chamber.html)</td><td></td></tr><tr><td>Solution for tissue preservation and nutrition&amp;nbsp;&amp;nbsp;</td><td>AL.CHI.MI.A. Srl, Viale Austria 14, 35020 - Ponte S. Nicol&amp;ograve; - PD&lt;br/&gt; &amp;nbsp;ITALY</td><td>Carry-C media for corneal deturgescence and transport at room temperature - 12 x 50 ml (http://www.alchimiasrl.com/en/organ-culture-at-31&amp;deg;-c-eb/carry-c-eb)</td><td></td></tr></tbody></table>

an &#8220;all-laser&#8221; endothelial transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

&quot;모든 레이저&quot;수술은 최소 침습 각막 이식을 수행하기 위해 제안된다. 대하여 표준 내피 이식, 각막 두께 측정 공여 조직을 염색 첨가 레이저 광 전달의 단계들 : 절차 (도 1 참조)을 수행하기 쉽다. 달성 이점은 주로 몇 분의 증가 수술 시간을 보상한다. 공여자 각막 두께와 도너 lenticule 치수를 지정하는 데 사용 펨토초 레이저를 측정하기 위해 수술 중의 OCT 사용 도너 / 호스트 ?...

Watch this Scientific Journal Video about An Ã¢â‚¬Å“All-laserÃ¢â‚¬Â Endothelial Transplant at JoVE.com

An &#8220;All-laser&#8221; Endothelial Transplant

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

&quot;모든 레이저&quot;내피 세포 이식

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

Research

JoVE Journal

Medicine

9.4K 조회수.  Italian National Research Council. An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

비디오: An “All-laser” Endothelial Transplant

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Autologous 내피 전구 세포 - 심는 기술 및 대형 동물 모델에서 순환기 장치에 대한 Biocompatibility 테스트

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

연구

생체공학

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

내피 Keratoplasty을위한 각막 기증자 조직 준비

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

의학

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Hindlimb 국소 빈혈의 치료를위한 배아 줄기 세포 유래 내피 세포

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

생물학

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

길이, 비 침습적을위한 안구의 앞쪽 상공 회의소에 이식<em&gt; 생체에</em실시간으로 단일 셀 해상도&gt; 영상

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet&#39;s membrane) is introduced onto the recipient&#39;s posterior stroma3. Unlike Descemet&#8217;s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity4. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision1. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet&#39;s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK5. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.

Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet&#39;s membranes from donor corneoscleral buttons for transplantation2.

데스 메막 내피 각막 이식을위한 각막 기증자의 조직 준비

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

공동 설계 된 내 피 세포와 인간 난소 조직 이식: 세포 기반 전략 결합 가속 직접 Paracrine 배달 관류

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

각막 내피 세포 손실의 비 침습적, 레이저 보조 실험 모델의 개발

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

조작된 섬유아세포 성장 인자 1에 의해 자극된 가속화된 치유를 통해서만 Descemet의 스트리핑의 인간 각막 배양 모델

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

지리적 위축의 눈이 큰 모델에 인간 배아 줄기 세포 유래 망막 색소 상피 세포의 subretinal 이식

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

내피에서 중간엽으로의 전환을 연구하기 위한 생체 내 심장 이식

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

저자 스포트라이트: 내피에서 중간엽으로의 전환 연구를 위한 신생아 이종성 쥐 심장 이식 모델  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

내피에서 중간엽으로의 전환 연구를 위한 신생아 이종성 쥐 심장 이식 모델

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...

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