지원 : NIDCD R01DC009801, P30DC010362, 5T32DC000040-17

윤리 정책이 프로토콜에서 인간의 종양 표본의 사용은 아이오와 기관 검토위원회의 대학 (IRB)의 승인을 받았다. 1. 설치 일 전에 종양의 수확 <ol><li> 외투 세포 배양 접시 : 멸균 문화 후드에서 뚜껑을 교체, 폴리스티렌 / 플라스틱 문화를 잘 / 접시의 바닥을 커버하기에 충분한 폴리-L-오르니 틴 (0.01 % 용액)을 추가하고, 적어도 상온에서 앉아 보자 2 시간. 유리 슬라이드 나 커버 슬립 촬상을 요구하는 실험 폴리스티렌에 대해 치환 될 수있다. </li><li> 2 시간 후, 흡입과 폴리-L-오르니 틴 솔루션을 제거하고 접시는 살균 문화 후드에서 완전히 건조 할 수 있습니다. 충분히 라미닌 솔루션 추가 (칼슘 10 ㎍ / ml의 + / 마그네슘 2 + 무료 HBSS [HBSS - / -]) 완전히 배양 플레이트를 커버하는, 뚜껑을 교체, 밤새 차가운 4 ° C 냉장고에있는 1) 장소 중 하나, 2) 최소 2 시간 동안 37 ° C 배양기에 배치합니다. 만약 판하룻밤 이상 저장됩니다, 증발 / 건조를 방지하기 위해 플라스틱 랩에 접시를 감싸줍니다. </li></ol> 2. 설치 종양 수확의 날 <ol><li> 준비 문화 미디어 : 미디어, 종양의 수확 및 가공의 날에 신선한 만든 4 ° C에 저장하고, 직전에 미디어 추가 / 변경을 인큐베이터에서 37 ° C로 가열된다. </li><li> 신경초종 문화 미디어 0.1 ㎎ / ㎖ 페니실린 및 0.1 ㎎ / ㎖ 스트렙토 마이신과 높은 포도당 (4.5 ㎎ / ㎖) (페놀 레드) DMEM입니다; N2 미디어는 최종 희석 1:100을 보충; 소 인슐린 5 ㎍ / ㎖의; 10 %의 총 부피에 태아 혈청. 0.22 μm의 필터를 통해 모든 미디어를 필터링 할 수 있습니다. </li><li> 표본 전송 냉각기를 준비 수술실에 2 + (HBSS + / +) 칼슘 2 + / Mg를 25 ㎖의 HBSS와 멸균 50 ML 원뿔 튜브 (수확을 종양의 크기에 따라) 1-2 얼음 가득 쿨러 보내기 종양 샘플 전송 및 수송. </li><li> 개별 선택을 준비idual 종양 해리 튜브 - 1 HBSS + / + ㎖의 얼음에 장소 라운드 멸균 2 ㎖ 아래 튜브를 입력합니다. (튜브는 종양 샘플의 날카로운 절개 / 해리에 사용될 것이다.) </li><li> 해리를 들면, 수확 된 조직의 매 0.5 cm 3 약 1 해리 튜브를 준비하고; 예를 들어, 1.0 × 1.0 × 1.0 cm (1cm 3) 측정 종양 표본이 HBSS + / + 해리 튜브를 필요로 할 것이다. </li><li> 자외선 살균 : 장소의 조직 가위, 작은 집게, 메스 핸들, 1X 일반 세균 배양 용 접시, P1000 피펫 및 멸균 문화 후드에 피펫 팁, ~에 자외선 아래에 남겨 이전의 종양 조직의 도착 및 처리에 15 분. </li></ol> 3. 검체의 분리 및 전송 <ol><li> 수술 적 절제에 의해 종양 표본을 분리합니다. </li><li> 즉시 환자에서 제거 후 가능한 한 빨리 얼음처럼 차가운 HBSS + / +에 종양 표본을 배치합니다. 일단 모든 Tissu은전자 샘플은 추가 처리를 위해 실험실로 수술 극장에서, (얼음)에 전송 샘플을 수집됩니다. </li></ol> 4. 조직 해리와 연화 <ol><li> 층류 후드 표준 무균 기술에 이어, 문화 후드에 종양 표본으로 원뿔 튜브를 가지고, 튜브 및 종양의 50 배를 반전시켜 샘플을 씻어. 조각이 튜브의 바닥에 떨어질하고 HBSS + / +를 제거 할 수 있습니다. 30 ㎖의 HBSS + / +로 보충하고, 교반 / 다시 50 배를 반전. 배의 총 반전 / 미디어 제거를 반복합니다. </li><li> 30 ㎖의 HBSS는 + / + 마지막으로, 다음 sterile100 mm 페트리 접시에 혼합물을 넣어 별도의 종양이 0.5 cm로 3 그룹화 종양 조각을 일시 중지 다시. 큰 종양 조각이 발생하는 경우, 작은 조각으로 잘라 조직 가위 또는 메스 (# 11, # 15 블레이드)를 사용합니다. </li><li> 개인 HBSS는 + / + 작성에 0.5 cm 3 조직 그룹을 배치 할 집게를 사용하여, 2 ㎖ 둥근 바닥 원뿔 곡선 (이차 곡선)모든 얼음에 알 튜브. &#39;Snowglobe에&#39;모양 (그림 1)이 있어야 서브 1mm 조각 - 현탁액에 종양 조각을 말하다 조직 가위를 사용합니다. 단편의 대부분이 서브 1mm 때까지만 종양 말하다; 하지 &#39;오버 말하다&#39;종양 샘플을한다. 다시 얼음에 완전히 다진 조직 관을 배치하고 모든 추가 표본 튜브 반복합니다. </li><li> 하나의 15 ML 원뿔 관에 모든 조각 종양 샘플을 전송합니다. 컷 / 수정 멸균 팁 P1000 피펫은이 단계에서 잘 작동합니다. 사용 추가 HBSS + / + 확인 모든 종양 샘플 15 ML 튜브에 수집 된 확인하기 위해 2 ㎖ 해리 튜브 린스 / 플러시. 5 분 73 XG에, 23 ° C에서 원심 분리기의 혼합물을 스핀 다운. </li><li> 세포 펠렛을 떠나, HBSS + / +를 흡입. 0.25 % 트립신의 1:1 혼합물에 조각을 다시하면 일시 중지 : (HBSS - /에 용해 -) - 0.2 % 콜라게나 종양 조각을 유지하기 위해 충분한 추가 꽉 스와spension. 30 분 동안 37 ° C의 배양기에서 나사 정상, 장소를 교체합니다. 현탁액 단편을 계속 배양하는 동안 적어도 한번 세포 혼합액을 교반한다. 뿐만 아니라이 시간에 따뜻하게하기 위해 인큐베이터에 신경초종 문화 미디어를 넣습니다. </li><li> 배양 후, 23 ° C, 5 분 73 XG에 트립신을 불 활성화하기 위해 각각의 튜브에 100 μL의 FBS를 추가 한 다음 샘플을 원심 분리기. 피펫을 사용하여 조심스럽게 세포 펠렛을 방해하지 않고 가능한 한 상층 액을 흡입. 1시 2분 종양 단편의 최종 비율 종양 단편에 가온 배양 배지 (N2/insulin/5 % 1 FBS)을 추가 배양 배지 (예, 종양 절편 1 ㎖가있는 경우에, 가온 2 ㎖에 쓰 미디어). </li><li> 약 1 ~ 2 mm의 직경에 P1000 피펫 팁 떨어져 끝을 절단하여 종양 분쇄를 준비합니다. 쉽게 unalt을 통해 솔루션을 피펫 할 수있을 때까지 천천히, 점진적으로 더 작은 직경의 피펫 팁을 사용하여 세포 현탁액을 씹다ered P1000 피펫 팁. 쉽게 팁을 통해 종양 솔루션을 피펫 수 있으면 즉시 작은 팁으로 이동 -에 샘플을 씹다가 없습니다. 원뿔 튜브의 바닥에 피펫 팁을 눌러 분쇄하는 동안 조직 블록 열망의 하나의 큰 조각은 종종 계속 분쇄을 허용하는 경우. </li></ol> 5. 셀 도금 <ol><li> 조직의 한 0.5 mm 3 조각은 1 개의 x 100mm 접시, 또는 4 개 8-well/4-well 슬라이드를 종자에 충분하다. 직전 미디어 / 세포 혼합물을 추가로 배양 접시에서 라미닌의 용액을 제거하지만 접시가 건조하게하지 않는다. </li><li> 각 100mm의 요리에, 10 ㎖ 따뜻하게 문화 미디어 종양 조각을 용해. </li><li> 4 - 웰 슬라이드의 각 웰의 경우, 750 ㎕를 따뜻하게 문화 미디어 (전체 슬라이드 3 ㎖)를 사용합니다. </li><li> 8 잘 슬라이드의 각 웰의 경우, 400 ㎕의 예열 문화 매체 (전체 슬라이드의 3.2 ML)을 사용합니다. </li><li> 37 &amp;의 문화를 품다# 160; ° C, 습도 100 %, 6 % CO 2. 적어도 36 시간 동안 혼자 문화를 그대로두고 용액 (산성) 황변 경우 미디어를 변경합니다. 그렇지 않으면 다음 2-3 일마다 또는 미디어가 산성이 될 때, 72 시간에 미디어를 변경합니다. 미디어가 연속 며칠 동안 매일 산성되면, 이것은 일반적으로 잠재적 인 세균이나 효모 오염의 표시이다. </li></ol>

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관심 없음 충돌은 공개 없습니다.

체외 신경초종 문화의 역사 체외 신경초종의 문화에서 설정하는 노력이 열려 수술 적 절제 1894 년 12 발생 초기 청신경 후 몇 년을 시작했다. 최초의 기록 문화가 실패 &quot;매달려 드롭 방법&quot;(12) 다진 종양의 성장을 시도 1920 년대에 Kredel로했다 . 나중에 박사 부부. 머레이와 스타우트는 응고 인간 혈장에서 성장 neurilemomas과...

전정 신경초종 (VSS는) 모든 두개 내 종양 1-3의 10 %를 손상 슈반 세포 전정 신경의 (SC) 종양을 나타냅니다. VSS는 모두 NF2 종양 억제의 비활성화 결함과 관련된 산발적 또는 가족 중 하나 (형 신경 섬유종증 (neurofibromatosis type 2), NF2) 형태의 발생 유전자. VS들에 대한 치료는 그러나 이러한 절차의 사망률은 청각 장애, 안면 신경 병증, 척수액 누출, 불균형, 그리고 종양의 재성장 1을 포함, 일반적으로 수술 적 절제 또는 방사선 수술입니다. 또한 모든 환자는 허용 수술 또는 방사선 후보입니다. 대체 요법의 이러한 중요한 합병증뿐만 아니라 부족은 새로운 치료 3,4 개발의 희망 VSS의 고유의 분자 생물학에 조사를 주도하고있다. 세포 배양은 잠재적 인 치료 컴의 분자 및 세포 행동과 심사의 신속 손쉬운, 심도있는 분석을 할 수 있습니다파운드. 역사적으로, 신선한 조직 표본에 대한 액세스 및 신경초종 세포는 불멸화되지 않는다는 사실의 부족이 크게 신경초종의 종양 형성의 연구에 대한 일차 배양의 사용을 방해했다. 차 문화의 공급이 제한을 극복하기 위해, 불후의 HEI193 VS 셀 라인은 HPV E6와 E7 유전자 (oncogene) 5와 전달에 의해 생성 된. 이 종양 전달은 인간의 신경초종 종양 모델로 사용을 제한하는 세포에 중요한 분자 및 표현형의 변화를 소개했다. 기능 NF2 유전자가 부족한 유전자 변형 마우스 라인에서 파생 된 SC 문화는 체외에서 NF2 의존 SC의 종양을 조사하기 위해 다른 대안을 나타냅니다. 이러한 문화는 그러나 인간의 특정 행동에 대한 인간 VS들 또는 계정의 이질적인 성격을 요점을 되풀이하지 못한다. 대부분의 이전 VS 배양 기술은 상대적으로 긴 처리 시간과 복잡한 선택적 배양 기술 6-8이 필요합니다.면역 염색에 의해 결정 여기서 우리는 95 %의 종양 세포의 순도 3 시간에서 완전 처리와 세포 배양 VS 차에 대한 간단한 재현 프로토콜을 제시한다.

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primary culture of human vestibular schwannomas

전정 신경초종 (VSS는) 모든 두개 내 종양의 10 %를 손상 슈반 세포 전정 신경의 (SC) 종양을 나타냅니다. VSS는 모두 NF2 종양 억제의 비활성화 결함과 관련된 산발적 또는 가족 중 하나 (형 신경 섬유종증 (neurofibromatosis type 2), NF2) 형태의 발생 유전자. VS들에 대한 치료는 그러나 이러한 절차의 합병증이 덜 침습적 인 치료로 조사를 주도하고있다, 일반적으로 수술 적 절제 또는 방사선 수술입니다. 역사적으로, 신선한 조직 표본에 대한 액세스 및 신경초종 세포는 불멸화되지 않는다는 사실의 부족이 크게 신경초종의 종양 형성의 연구에 대한 일차 배양의 사용을 방해했다. 차 문화의 공급이 제한을 극복하기 위해, 불후의 HEI193 VS 셀 라인은 HPV E6와 E7 발암 유전자와 형질 도입에 의해 생성 된. 이 종양 전달은 인간의 모델로 사용을 제한하는 세포에 중요한 분자 및 형질 변경을 도입chwannoma 종양. 따라서 우리는 세포 VS 인간의 기본 문화에 대한 단순화, 재생 가능한 프로토콜을 보여줍니다. 이 간단 익힌 기술은 더 정확하게 VS 질환의 복잡성을 다시 요약 분자 및 세포 조사를 허용한다.

정확한 종양의 분열은 조직 배양 접시에서 최적의 파종 및 파생물 (그림 1)을 위해 필수적입니다. 도금 후의 제 일 동안 신경초종 셀 식별 인해 세포 파편 및 적혈구의 양을 불명료에 종종 어렵다. 4 번째 또는 5 번째 날에 의해, 부착 종양 조각 근처 세포 확장되는 파편 패턴을 &#39;몰&#39;인식 만듭니다. 이후 미디어의 변화는 일반적으로 두 번째 주에 비 부착 세포의 대?...

Watch this Scientific Journal Video about Primary Culture of Human Vestibular Schwannomas at JoVE.com

Primary Culture of Human Vestibular Schwannomas

전정 신경초종 (VSS는) NF2 종양 억제 유전자의 돌연변이와 관련된 슈반 세포 (SC) 기원의 비 악성 종양이다. 우리는 분자 및 세포 실험 조작 및 분석이 가능하며 인간의 질병의 이질적인 성격을 되풀이되었습니다 세포 배양 VS 인간의 기본에 대한 재현, 효율적인 프로토콜을보고합니다.

인간의 전정 신경초종의 차 문화

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

primary-culture-of-human-vestibular-schwannomas

Research

JoVE Journal

Medicine

12.7K 조회수.  University of Iowa Hospitals and Clinics.  전정 신경초종 (VSS는) NF2 종양 억제 유전자의 돌연변이와 관련된 슈반 세포 (SC) 기원의 비 악성 종양이다. 우리는 분자 및 세포 실험 조작 및 분석이 가능하며 인간의 질병의 이질적인 성격을 되풀이되었습니다 세포 배양 VS 인간의 기본에 대한 재현, 효율적인 프로토콜을보고합니다. 

비디오: Primary Culture of Human Vestibular Schwannomas

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

인간의 기본 섬유 아세포의 유도를위한 피부 펀치 생검 절편 문화

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

연구

의학

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

기본 마우스 대장 종양의 체외 Organoid의 문화

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

인간의 태아 췌장 세포 클러스터의 분리, 문화, 및 이미징

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

차 사람 골수 및 악성 종양을 연구하는 세 가지 차원 조직 배양 모델

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

인간의 뇌하수체 선종 세포의 3 차원 알긴산 비드 문화

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

개 동맥과 정맥에서 차 내피 세포의 분리 및 문화

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

기간 Placentae의 태아 세포 막에서 기본 인간 Decidual 세포의 고립

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

근육의 불균형: 테스트 및 기능 편심 햄 스트링 운동 인구 강도 훈련

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

Vestibular 시스템 공부를 쥐의 외과 Labyrinthectomy

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

소설 인간 오르가노피 망막 문화 기술의 특성화

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...