We wish to thank Alison Dorsey for helping to develop the subretinal injection technique. We also acknowledge the National Eye Institute (NEI grants EY11254 and EY021416), California Institute for Regenerative Medicine (CIRM grant TR1-01219), and the Lowy Medical Research Institute (LMRI) for very generous funding for this project.

참고 : 모든 동물이 스크립스 연구소에 의해 설립 된 윤리 지침에 따라 처리 하였다. 사출 용 재료 1. 준비 (~ 20 분) <ol style="margin-left: 40px;"><li> 사전 따뜻한 세포 분리 솔루션 (바람직하게는 혈청 희석을 통해 비활성화되어 하나), 멸균 PBS, 문화 미디어 (표 1). </li><li> 그것을 분해하고 15 분 동안 물에 부품을 끓여 무딘 바늘과 주사기를 소독. </li></ol> 사출에 대한 RPE 세포의 2. 준비 (~ 30 분 내지 1) <ol style="margin-left: 40px;"><li> 37 ℃에서 5-8 분 동안 예열 세포 분리 용액을 사용하여 RPE 세포를 분리. </li><li> 여전히 연결되어 있는지 어떤을 해제 부드럽게 세포를 긁어. </li><li> 배양액 대용량 (15ml의 튜브를 기입) 해리 용액을 비활성화하고 카운트하기로 세포를 희석. </li><li> 펠릿 세포 5 분 동안 800 XG에 원심 분리기. </li><li> 멸균 미리 예열 PBS에서 / μL 200,000 세포 (0.5 μL 볼륨에서 10 만 셀을 제공하기 위해)에서 세포를 재현 탁하고 1.5 ml의 마이크로 원심 튜브로 전송합니다. </li><li> 선택적으로, 살아있는 세포 과도 형광 마커를 추가하고 30 ~ 45 분 동안 37 ℃에서 배양한다. </li><li> 세포의 0.5 μL와 무딘 바늘과 주사기를로드합니다. 가능한 빨리 세포 주입한다. </li></ol> 3. 하위 망막 사출 (~ 사출 당 5 분) 참고 : 가능하면 윤부 선박 시각화 훨씬 용이하기 때문에, 성인 흰쥐와 기술을 배웁니다. 보다 쉽게​​ 주사 부위의 시각화를 용이하게하기 위해 (세포를 주입하기 전에) 배울 때 빠른 그린 용액을 주입한다. <ol style="margin-left: 40px;"><li> 쥐를 마취. 100 ㎎ / ㎖ 케타민 10 ㎎ / ㎖ 자일 라진 (xylazine) (20 μL / 10g 본체 w의 복강 내 주사를 사용하여이소 플루오 란 흡입을 통해 여덟)는 설치류를 기동 흡입기의 주둥이와 눈에 주입하기 어렵 기 때문에. <ol><li> 동물 깊이 그것의 발 중 하나를 집어 마취되어 있는지 확인합니다. 이 flinches 경우, 몇 분 이상 기다린 후 망막 주입을 시작하기 전에 다시 시도하십시오. </li></ol></li><li> 주입되는 눈이 천장을 향하도록 옆에 쥐를 놓습니다. </li><li> 눈 소켓 (임시 안구 돌출)에서 튕겨 나올 그냥 귀 위의 턱으로 가볍게 눈꺼풀에 피부 평행 스트레칭 두 손가락으로 머리를 잡고 더 접근하기가되도록 해부 현미경 부드럽게 피부를 스트레칭 그래서 눈이 약간 소켓에서 팝업됩니다 (그림 1C 참조). 목에 너무 가까이 쥐를 잡지 마십시오. </li><li> 30 G 날카로운 일회용 사전 멸균 바늘, 선박이 충돌하는 경우, 출혈입니다 b (즉시 윤부 아래에 구멍을중요한 전자와 나중에 구멍을 찾기 어려울 수)과 각도로 바늘 (그림 1D)으로 렌즈에 손이 닿지 않도록. 발생 날카로​​운 (또는 무딘) 바늘 또는 즉시 백내장으로 렌즈를 만지지 마십시오. 참고 : 주사는 두 사람이 잘 작동. 이런 식으로 한 사람은 구멍이 어디에 초점을 유지하기 위해 날카로운 일회용 바늘로 첫 번째 구멍을 만든 후 주입 작업을하는 사람이 무딘 바늘 주사기를 전달할 수 있습니다. </li><li> 머리에 그립을 유지하면서 눈에서 일회용 날카로운 바늘을 집어 넣으십시오. 구멍이 정확히 기억하십시오. </li><li> 중 미세 조작기에 무딘 바늘로 사전로드 된 주사기를 장착하거나 손을 잡고, 렌즈를 만지지 조심스럽게 눈을 통해 밀어 다시는주의하면서 구멍을 통해 무딘 바늘 주사기의 끝 부분을 삽입 한 후 아주 부드럽게 느낌 저항 (그림 1D)까지. </li><li> 케이최소한으로 모든 움직임을 eeping, 조심스럽게 천천히 망막 하 공간에 RPE 세포를 주입. 참고 : RPE / 망막 박리가 유도 될 것이다; 이 불가피하다. 그러나, 청소기 주입 박리를 최소화 크게 재 부착 (도 1E)의 기회를 향상시킨다. 모든 과장된 움직임은 망막에 다시 바늘을 이동 할 수 있으며, 옆으로 움직임이 망막에 손상을 줄 수 있습니다. 분사 펌프의 사용은 선택적이지만, 매우 정확한 전달을 허용한다. </li><li> 천천히 주사기를 집어 넣으십시오. 눈 보습을 적용하는 것은 수화 눈을 떼지 삭제합니다. </li><li> 이 흉골 드러 누움을 회복 할 때까지 동물을 모니터링하기 위해 계속합니다. 무인 동물을 떠나거나 흉골 드러 누움을 회복 할 때까지 다른 경고 동물 케이지에 반환하지 않습니다. </li></ol>

None of the authors have any commercial disclosures to declare.

이 문서에서 우리는 쥐와 생쥐에서 부유 망막 색소 상피 세포의 망막 주사를 수행하기위한 비교적 간단한 방법을 설명합니다. 이 프로토콜은 적은 외상에 번역 할 기술로 쉽게 배울 수 많은 경험이다 (그림 3, 이것은 더 나은 주사 중 하나를 나타냅니다) 미세 조작기는 (그림 1A)를 사용하는 경우에는 특히. 모든 외상은 cSLO 간섭 단층 시스템 (그림 2) 가능한 경?...

The sensory retina is organized in functional tiers of neurons, glia, and endothelial cells. Photoreceptors at the back of the retina are activated by light; through phototransduction they convert photons into electrical signals that are refined by interneurons and transmitted to the visual cortex in the brain. Phototransduction cannot occur without the coordinated efforts of Mueller glia and retinal pigment epithelium (RPE) cells. RPE are organized in a monolayer directly behind the photoreceptors and perform multiple and diverse functions integral to photoreceptor function and homeostasis. In fact, RPE and photoreceptors are so co-dependent that they are considered to be one functional unit. Death or dysfunction of RPE results in devastating secondary effects on photoreceptors and is associated with age-related macular degeneration (AMD), the leading cause of blindness in the elderly1,2.
While no cure has been discovered for AMD, several clinical studies have shown that RPE cell replacement may be a promising therapeutic option3-13. With the advent of stem cell technology, it is now possible to generate large numbers of RPE cells in vitro from embryonic and induced pluripotent stem cells (hES and hiPS) that strongly resemble their somatic counterparts functionally and anatomically14-26. Stem cell-derived RPE have also been shown to function in vivo by multiple independent groups, including our own, to significantly slow retinal degeneration in rat and mouse lines with spontaneous retinal degeneration16,18,21,22,25,28,29. This combination of clinical and preclinical supporting evidence is so compelling that several clinical trials to prevent retinal degeneration using stem cell-derived RPE cells are now ongoing30,31. 
RPE can be readily derived from hES and/or hiPS and implanted in the subretinal space of rodents using various derivation and injection techniques32,33. (See Westenskow et al. for a methods paper in multimedia format demonstrating the directed differentiation protocol we employ)34. There are critical remaining questions regarding the safety, survival, and functional capacity of exogenously delivered RPE cells upon implantation, therefore the ability to perform subretinal injections in rodents is a critical skill16,18,21,29,36,37. The delivery of RPE is not trivial, and the field is divided on the most effective injection technique. The protocol we describe here is a simple and effective way to deliver of bolus of RPE cells subretinally, and was used in the first clinical trial for stem cell-derived RPE transplantation31. (The reader may also refer to another JoVE article by Eberle et al. for an alternative depiction of subretinal injections in rodents.38) 
The technique outlined in this manuscript cannot be visualized and trauma is unavoidable (as with any subretinal injection technique). It is performed by making a hole just under the limbus vessels and inserting a blunt needle along a transscleral route to inject a bolus of cells under the diametrically opposed retina. The person doing the injection will feel resistance as the blunt needle touches the retina. The cells may be directly visualized after the injection, however, and the degree of the induced retinal detachment can be determined by labeling the RPE cells with a transient fluorescent marker and detecting them with a confocal scanning ophthalmoscope (cSLO). An optical coherence tomography (OCT) system can also be used to monitor the trauma and easily identify the injection site.

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:419px;">Name of Material/ Equipment (A-Z)</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-Mercaptoethanol (55 mM)</td>
			<td>Gibco&#160;</td>
			<td>21985-023</td>
			<td>50 mL x 1&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Scapers</td>
			<td>VWR</td>
			<td>89260-222</td>
			<td>Case x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CellTracker Green CMFDA</td>
			<td>Molecular Probes</td>
			<td>C34552</td>
			<td>50 ug x 20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>500 mL x 1&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fast Green</td>
			<td>Sigma-Aldrich</td>
			<td>F7258</td>
			<td>25 g x 1&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Genteal Geldrops Moderate to Severe Lubricant Eye Drops&#160;</td>
			<td>Walmart</td>
			<td>4060941</td>
			<td>25 mL x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hamilton Model 62 RN SYR</td>
			<td>Hamilton</td>
			<td>87942</td>
			<td>Syringe x 1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Hamilton Needle 33 gauge, 0.5&#34;, point 3 (304 stainless steel)</td>
			<td style="width:145px;">Hamilton</td>
			<td style="width:119px;">7803-05</td>
			<td>Needles x 6</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Knockout DMEM</td>
			<td>Gibco</td>
			<td>10829-018</td>
			<td>500 mL x 1&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KnockOut Serum Replacement</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td>500 mL x 1&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine 200 mM</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:419px;">Magnetic Stand</td>
			<td style="width:145px;">Leica Biosystems</td>
			<td style="width:119px;">39430216</td>
			<td>Stand x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MEM Non-Essential Amino Acids Solution 100X&#160;</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:419px;">Micromanipulator</td>
			<td style="width:145px;">Leica Biosystems</td>
			<td style="width:119px;">3943001</td>
			<td>Manipulator x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100 mL x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slip Tip Syringes without Needles BD&#160; (3 mL)&#160;&#160;</td>
			<td>VWR</td>
			<td>BD309656</td>
			<td>Pack x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specialty-Use Needles BD&#160; (30 gauge, 1&#34;)</td>
			<td>VWR</td>
			<td>BD305128</td>
			<td>Box x 1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE Express Enzyme (1X), no phenol red</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td>100 mL x 1</td>
		</tr>
	</tbody>
</table>

performing subretinal injections in rodents to deliver retinal pigment epithelium cells in suspension

전기 자극으로 광 변환 외측 망막에서 발생 콘로드와 ​​감광체와 망막 색소 상피 (RPE) 세포에 의해 주로 수행된다. RPE는 감광체 및 사망 또는 RPE 세포의 기능 장애 연령 관련 황반 변성 (AMD), 명 55 세 이상에서 영구 실명의 주요 원인의 중요 특성 인 지원을 제공한다. AMD에 대한 치료법이 발견되지 않은 반면, 병변 RPE 건강한 눈 주입은 효과적인 치료를 증명할 수 있고, RPE 세포의 다수 용이 다 능성 줄기 세포로부터 생성 될 수있다. RPE 세포 전달의 안전성과 효능에 관한 몇 가지 흥미로운 질문 여전히 동물 모델에서 시험 될 수 있고, RPE를 주입하기 위해 사용되는 잘 용인 프로토콜이 개발되었다. 여기에 기재된 기술은 다양한 여러 연구 그룹에 의해 사용 된과 제 날카로운 바늘의 눈 구멍을 만드는 것을 포함한다. 블루와 다음 주사기세포로드 NT 바늘 구멍을 통해 삽입하고 부드럽게 RPE에 닿을 때까지 유리체를 통과한다. 비교적 간단하며 최소한의 설비를 필요이 주입 방법을 사용하여, 우리는 동물 모델에서 광 수용체 변성증의 상당량을 방지 호스트 RPE 사이에서 줄기 세포 유래 RPE 세포를 효율적이고 일관성있게 통합을 달성. 실제 프로토콜의 일부 있지만, 우리는 또한 주입하는 방법과 세포가 생체 내 이미징을에 사용하여 망막 하 공간에 주입되었는지 확인하는 방법에 의해 유도 된 외상의 범위를 결정하는 방법에 대해 설명합니다. 마지막으로,이 프로토콜의 사용은 RPE 세포에 한정되지 않는다; 이는 망막 하 공간에 임의의 화합물 또는 세포를 주입하는 데에 이용 될 수있다.

우리는이 논문에서 기재된 기술을 사용하여 신속하고 지속적으로 설치류 망막 하 공간 내로 RPE 세포의 현탁액을 제공 할 수있다. 필요한 것은 아니지만, 외상은 크게 그림 1A &amp; B의 미세 조작기와 같은 설정을 사용하여 최소화 할 수 있습니다. 일시적인 안구 돌출 그림 1C에서와 같이 쥐를 잡으십시오. 미세 조작기 또는 손에 의해 수행되는 경우에 단계는 동일; 이 ...

Watch this Scientific Journal Video about Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension at JoVE.com

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

서스펜션에 망막 색소 상피 세포를 제공하기 위해 설치류에서 망막 주사를 수행

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

performing-subretinal-injections-rodents-to-deliver-retinal-pigment

Research

JoVE Journal

Medicine

17.2K 조회수.  The Scripps Research Institute. Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

비디오: Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

같은 자폐증과 정신 분열증과 같은 일반적인 장애에 드 노보 변이를 식별하는 전략

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

연구

의학

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

마우스 눈에 유전자 치료 벡터와 줄기 세포의 Subretinal 주입

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

나트륨 히알루 론산의 망막 주사 설치류에있는 망막 박리 모델

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

토끼의 망막 수술에 대한 단계 프로토콜에 의해 단계

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

인간 종양 이종 이식에서 마우스 세포의 고갈은 크게 대상 세포의 다운 스트림 분석을 향상

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the Norwood-Hamilton classification scale, and female pattern hair loss (FPHL) in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with non-activated autologous platelet-rich plasma (A-PRP). Autologous blood (55 mL) was harvested using sodium citrate as an anticoagulant. A-PRP (23 mL) was produced for all cases using a closed system according to the transfusion service protocol. Following centrifugation (260 x g for 10 min) the A-PRP was inserted in a laser light selector device, and after the centrifugation, 9 mL of A-PRP was collected.
The scalp of the patients affected by androgenetic alopecia (AGA) was divided into four areas (frontal, parietal, vertex, and occipital); local anesthesia was not performed. Interfollicular A-PRP injections (0.2 mL x cm2) were performed by controlled and mechanical injections scheduled at a depth of 5 mm using a medical injector gun. Treatment sessions were performed with a 30-day interval. For each patient, three treatment sessions were performed.
PRP was injected in the androgen-related areas of scalp affected by hair loss. Placebo (normal saline solution) was loaded in another syringe (10 mL) and injected on the adjacent side in a similar fashion.

This paper aims to describe the interfollicular autologous platelet-rich plasma injections (0.20 mL x cm2) in the scalp of 23 patients affected by androgenetic alopecia at a depth of 5 mm using a medical injector gun equipped with a 30 gauge needle, in three sessions.

Androgenetic 탈모 증에 의해 영향을 받는 환자에서 기계 및 제어 PRP 주사

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the ...

Using a Chemical Biopsy for Graft Quality Assessment

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient&#8217;s life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient&#8217;s body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

The protocol presents utilization of the chemical biopsy approach followed by comprehensive metabolomic and lipidomic analysis for quality assessment of kidney grafts allocated for transplantation.

이식 품질 평가를 위해 화학 생검 사용

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

설치류에 있는 허혈성 망막 질병을 위한 산소 유도한 망막병증 모형

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

수면 의 아름다움 트랜스포슨 - 전염 인간 망막 안료 상피 세포에서 산화 스트레스의 유도 및 분석

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...