이 작품은 (HPL에) NIH 보조금 R01 GM087609, (MTL에) (HPL에) 앤서니 슈, NIH 보조금 U01의 HL099776의 명예에 잉그리드 라이 빌 슈의 선물, Hagey 실험실에서 보조금에 의해 부분적으로 지원 소아 재생 의학 및 오크 재단 (MTL에, GCG와 HPL). GGW 의학의 스탠포드 대학, 스탠포드 의료 과학자 훈련 프로그램에 의해 지원 및 NIGMS 훈련 보조금 GM07365했다. ZNM는 성형 외과 재단 연구 활동 그랜트와 Hagey 가족 기금에 의해 지원되었다. MSH는 캘리포니아 재생 의학 연구소 (CIRM) 임상 연구원 교육 교부금 TG2-01159, 악안면 외과 의사의 미국 사회 (ASMS) / 악안면 외과 재단 (MSF) 연구 그랜트 수상하고, 이식 및 조직 공학 원정대 수상에 의해 지원되었다.

이 프로토콜은 실험 동물 관리에 스탠포드 대학 행정 패널에 의해 승인 된 방법을 따른다. 쥐 진피 1. 소화 <ol><li> 케타민 100 ㎎ / ㎏ + 자일 라진 (20) ㎎ / ㎏ + 아세 프로 마진 3 ㎎ / ㎏의 복강 내 주사하여 마취 후 경추 탈구로 쥐를 안락사. 참고 : 다양한 연령과 배경을 사용할 수 있습니다. </li><li> 면도와 지느러미 피부를 털을 뽑다. 약 100,000 세포 등쪽 피부 조각 X 100mm 60mm로부터 분리 될 수있다. </li><li> 깨끗한 건조 멸균 표면에 70 % 에탄올 대신에 마우스를 담근다. </li><li> 즉시 멸균 해부 가위를 사용하는 등의 마우스 피부를 수확. 암컷 마우스에서 유방 조직을 포함한 피. </li><li> 꼬리의 기초부터 텐트까지 피부에 집게를 사용하여 문헌-근막면을 따라 절개하기 전에 가로 절단을합니다. </li><li> 조심스럽게 수확하는 동안 어떤 피하 지방을 포함하지 않도록피부. 어떤 피하 지방의 수확 피부를 검사하고 조심스럽게 메스의 무딘 가장자리를 사용하여 긁어. </li><li> 얼음에 PBS 세척을 5 배 뒤에 betadine에서 수확 피부를 씻어. 주 : 오염을 방지하기 위해 가능한 무균에 가까운 피부를 유지하는 것이 중요하다. </li><li> 면도날을 사용하여 샘​​플이 2-3mm 조각 균일 일관성 때까지 멸균 접시에 가위를 해부 피부를 말하다. </li><li> DMEM에 1 ㎎ / ㎖의 농도로 50 ml의 20 ㎖의 콜라게나 제 IV를 함유하는 원뿔형 튜브를 준비한다. 튜브 당 다섯 생쥐 기초 튜브로 진피를 나눈다. </li><li> 수조 나 오븐 중 1 시간 동안 37 ℃에서 배양하는 동안 샘플을 격렬하게 교반한다. </li><li> 인큐베이터에서 샘플을 제거하고 멸균 후드에 바늘 3 ~ 5 배없이 10 ML의 주사기를 통해 전달합니다. </li><li> 37 ° C에서 다시 인큐베이터에 샘플을 놓고 추가로 30 분 동안 격렬하게 흔들어. </li><li> 멸균 후드, 10 ml의 피펫을 사용하여 위 3 ~ 5 배 아래 샘플을 피펫. 새로운 50 ML 원뿔 튜브에 100 μm의 필터를 통해 샘플을 피펫. </li><li> 세포의 수율을 최대화하고 40 ml의 총 부피를 가지고 동일한 필터를 10 % FBS DMEM 20㎖를 전달한다. 4 ℃에서 8 분 300g에서 원심 분리기. </li><li> 먼저 뜨는 나머지 이전에 상부 지방층을 제거하기 위해 큰 관심을 가지고, 멸균 유리 피펫을 이용하여 상층 액을 제거합니다. 참고 :이 단계는 지방 세포의 오염을 감소하는 것이 중요합니다. </li><li> 20 ml의 10 % FBS DMEM으로 펠렛을 재현 탁. </li><li> 70 μm의 필터를 통해 세포 / DMEM 서스펜션을 전달합니다. </li><li> 4 ℃에서 8 분 동안 300g에 10 ml의 10 % FBS DMEM과 필터 원심 분리기 필터링 된 현탁액을 씻어. </li><li> 다시 처음으로 남아있는 지방 층을 제거하기 위해주의하면서 멸균 유리 피펫을 이용하여 상층 액을 제거합니다. </li><li> RBC 상당한 오염이있는 경우 (펠릿 가시적 적색), 펠릿을 다시 일시20 mL의 ACK 용해 완충액 RT에서 5 분 동안 배양한다. 그렇지 않으면 24 단계로 건너 뜁니다. </li><li> FACS 완충액 (PBS, 10 % FBS, 0.1 % 아 지드 화 나트륨)의 동일 부피 (20 ㎖)을 첨가 한 후 혼합하고 흠없는 대조군으로 제쳐 5 ml의 분취 량을 유지한다. 4 ℃에서 8 분 동안 300g에 남아있는 샘플을 원심 분리기. </li><li> 상층 액을 제거하고 얼음에 펠렛을 넣어. FACS 시간을 사용할 수없는 경우 세포는이 시점에서 내려 고정 될 수있다. </li></ol> FACS에 의한 섬유 아세포 2. 분리 <ol><li> 각각의 펠릿에 대한 계보 항체 배양 믹스 500 μl를 확인합니다. , CD45 (1 : 200), Tie2의 (1시 50분), 테르을 : 먼저 튜브에 DNase의 (10 μg의 / ㎖)를 포함 외과 버퍼의 475 μl를 추가 한 다음 형광 접합 CD31 (100 1)를 추가하여이 작업을 수행 -119 (1 : 200), 및는 EpCAM (1 : 100)은 각각의 항체에 대한 항체를 각 희석액을 달성한다. </li><li> 리니지 항체 배양 믹스 500 μL의 각 펠렛을 다시 중단하고 20 분 동안 얼음이 현탁액을 품어. </li><li> 5 추가샘플의 DNase (10 μg의 / ㎖)를 포함하고 부드럽게 혼합 ml의 외과 버퍼. 4 ℃에서 8 분 300g에서 원심 분리기. </li><li> 상등액을 제거하고, 단계 (26)에서와 같이 5 ㎖의 FACS DNase를 함유 완충액 (10 μg의 / ml) 및 동일한 조건을 이용하여 원심 분리 세포 펠렛을 세척 하였다. </li><li> DNase의 (10 μg의 / ㎖)를 포함 500 μL FACS 버퍼에 펠렛을 재현 탁하고 생존 염료 컨트롤로 옆으로 50 μL 나누어지는했습니다. </li><li> 선택한 염료에 대한 표시 농도의 나머지 샘플 선택의 생존 염료를 추가합니다. </li><li> FACS 분석 (31)을 수행하고 생존 염료 - / CD31- / CD45- / Tie2- / 테르 - 119- / EpCAM- 세포 (그림 2A 참조) 정렬. 정렬 직접 FACS 버퍼에. </li></ol>

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<li>Biburger, M., Trenkwald, I., Nimmerjahn, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((yThree+blocks+are+not+enough+-+Blocking+of+the+murine+IgG+receptor+FcgammaRIV+is+crucial+for+proper+characterization+of+cells+by+FACS+analysis%5BTitle%5D)+AND+%22European+journal+of+immunology%22%5BJournal%5D)">yThree blocks are not enough - Blocking of the murine IgG receptor FcgammaRIV is crucial for proper characterization of cells by FACS analysis.</a> European journal of immunology. , (2015).</li>
</ol>

The authors have nothing to disclose.

이 논문에 설명 된 프로토콜이 하위 집단을 위해 선택 또는 후속 분석하기 전에 세포 배양에서의 시간이 필요하거나 기존의 방법에 비해, FACS 기반 정렬하여 섬유 아세포를 분리하는 수단을 제공한다. 섬유 아세포의 선별을 피부로부터 채취 필요한 시간은 약 6 시간이고; 그러나, 수확에 사용 된 마우스의 수는이 추정에 영향을 미칠 것이다. 프로토콜의 몇 가지 포인트는 특?...

섬유 아세포는 자주 플라스틱 기판에 부착 스핀들 모양의 세포로 형태 학적으로 정의된다. 합성 섬유 아세포 및 배아 및 성체 기관 (1)의 세포 외 매트릭스를 리모델링을 담당 원리 세포 유형이다. 섬유 아세포는 포유류의 개발에 중요하며, 따라서 각 조직 및 기관에 존재하는 세포 유형 이웃의 동작에 영향을 미치는 세포 외 환경에 실질적으로 기여한다. 섬유 아세포는 엄청난 임상 부담의 원인이 의료 조건의 다양한 세트 뒤에 주요 세포 유형입니다. 병리학 섬유 아세포 활성은 정상 조직의 기능 손상 및 피부 상처 치유, 죽상 동맥 경화증, 전신성 경화증, 혈관 손상 2-5 후 아테롬성 플라크의 형성 다음 조직 (예 : 폐, 간 등) 장기 섬유증, 반흔을 포함한다. 특히 상처 치유, 모두 급성과 만성, D를 포함정상 조직 등에도 유사한 기능이나 그것을 둘러싼, 다양한 병리학 적 상태에 걸쳐 상당한 이환율을 초래 흉터 조직의 eposition. 손상 후,이어서, 구조적 ECM 성분을 분비하는 세포 유형에 인접한 주변 분비 효과를 발휘하고, 흉터 조직 (6)을 증착하여 기계적 안정성을 복원 근섬유 아세포로의 전환이있다. 피부 조직에서 발달 시간에 걸쳐 감겨 수리의 품질 및 해부학 적 부위 사이에 상당한 변화가 존재한다. 인생의 첫 두 학기에 태아는 흉터없이 치유; 그러나,에 성인기에 걸쳐 세 번째 임신에서, 상처와 치유 인간. 사이트 별, 연령별 이외에, 상처 치유의 차이가 존재한다. 피부의 상처 흉터 조직 내의 증착 9 상당한 동안 구강에 상처, 최소 반흔 7,8으로 개조. 논쟁은 C를 지속연령 및 위치 10, 11 모두에 관해서 상처 치유의 결과를 로컬 섬유 아세포의 고유 특성에 비해 환경의 영향을 상대적 oncerning. 피부의 진피 및 이전 배아 (E15) 대 구강 마우스의 치료에 유의 한 차이를 감안할 대 이후 배아 (E18) 진피, 그것은 특정 발달 연령에 다양한 해부학 적 부위 중 섬유 아세포의 인구에 본질적인 차이가 존재하는 것 같다 . 1986 년 해롤드 F. 드보락은 종양이 12을 치유하지 않는 상처입니다 상정. 드보락은 종양이 신체의 상처처럼 동작하고 호스트의 응답 상처 치유를 활성화시킴으로써 그들의 기질을 유도한다는 결론을 내렸다. 많은 연구 이후 암 13-15의 진행에 섬유 아 세포의 기여를 조사하지만, 한 상처 치유, 정체성과 피부 캘리포니아의 기질 구획에 기여하는 섬유 아 세포의 배아 기원의 경우와 같이rcinomas 적절하게 정의되지 않았습니다. 이 질문에 대한 대답은 항암 치료 (16)에 대한 잠재적으로 효과적인 대상으로 종양 관련 섬유 아세포를 노출 최근의 연구 주어진 의료 관련을 맺는다. 식별 및 전향 생체 폐의 섬유 전위 부여 섬유 아세포 계통을 분리하는 것은 효과적으로 급성 및 만성 질환 상태의 넓은 범위에 걸쳐 손상에 대한 반응을 조작 향해 필수적인 단계이다. 1987 년 Cormack는 유두와 망상 진피 (17, 18) 내에서 하나의 내부에있는 섬유 아세포의 두 개체군, 하나를 보여 주었다. 세 번째 모집단은 여포 (19, 20)의 모유두 지역에서 모낭과 관련된 발견되었다. 때 배양, 성장 잠재력, 형태, 및 성장 인자 / 사이토 카인 이러한 섬유 아세포 아형 전시 차이는 21 ~ 24 프로필. 현재까지, 섬유 아세포를 조사 연구 그는terogeneity 크게 적절하게 생체 내에서 섬유 아세포 중 발달 및 기능 다양성의 특성을 실패했습니다. 이것은, 부분적으로, 배양 된 섬유 아세포의 집단에 의존하고, 모든 섬유 아세포 (25)에 의해 표현되는 자기 표면 수용체의 기초 하에서 세포 배양 또는 양성 선별의 균질화 효과의 결과이다. 우리의 실험실에서 최근의 연구는 깊은 표면 마커이 원고 (26)에 제시된 FACS 기반의 분리 방법에 의해 분리 대 교양 교양 섬유 아 세포에서 전사 변화를 보여 주었다. 그 후, 우리는 쥐의 등쪽 진피 내의 특정 섬유 아세포의 계보를 파악하고 Engrailed-1의 배아 식으로 정의이 계보는,, 등의 피부의 결합 조직 증착을위한 일차적 책임이 있다고 결정했다. 상처 치유, 암 기질 형성 등을 포함 섬유증의 급성 및 만성 형태 모두 동안의 계보 기능방사선 섬유증 (27)을 유도. 별개의 섬유 아세포 계통의 특성은 폐의 섬유 성 행동을 조절하기위한 치료를위한 중요한 의미를 가지고있다. 오히려 세포 격리 28, 29을 달성하기 위해 체외 조작에 의존하는 기존의 프로토콜을 사용하는 것보다, 여기에 설명 된 수확 프로토콜 (그림 1)보다 정확하게 생체 내에서 표현형과 동작을 캡처 섬유 아 세포의 수율 정보 분석을하는 데 도움이됩니다.

<table><tbody><tr><td>Surgical Forceps</td><td>Kent Scientific</td><td>INS650916</td><td></td></tr><tr><td>Micro-scissors</td><td>Kent Scientific</td><td>INS600127</td><td></td></tr><tr><td>Povidone Iodine Prep Solution</td><td>Dynarex</td><td>1415</td><td></td></tr><tr><td>Nair (depilatory cream)</td><td>Church and Dwight Co.</td><td>22600267058</td><td></td></tr><tr><td>Collagenase IV</td><td>Gibco</td><td>17104-019</td><td></td></tr><tr><td>Elastase</td><td>Abcam</td><td>ab95133</td><td></td></tr><tr><td>DMEM</td><td>Life Technologies</td><td>A14430-01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) lysing buffer</td><td>Gibco</td><td>A10492-01</td><td></td></tr><tr><td>40 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-1</td><td></td></tr><tr><td>70 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-2</td><td></td></tr><tr><td>100 &amp;mu;m filters</td><td>Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>CD31</td><td>BioLegend</td><td>102421</td><td></td></tr><tr><td>CD45</td><td>BioLegend</td><td>103125</td><td></td></tr><tr><td>Tie2</td><td>BioLegend</td><td>124005</td><td></td></tr><tr><td>Ter-119</td><td>BioLegend</td><td>116233</td><td></td></tr><tr><td>EpCAM (CD326)</td><td>eBioscience</td><td>48-5791</td><td></td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>propidium iodide (PI viability stain)</td><td>BioLegend</td><td>421301</td><td></td></tr></tbody></table>

murine dermal fibroblast isolation by facs

섬유 아세포는 세포 외 기질을 분비에 대한 책임 원칙 세포의 유형과 여러 장기와 조직의 중요한 구성 요소입니다. 섬유 아세포의 생리와 병리 여러 기관에서 fibroses, 비후성 흉터 다음 화상, 허혈 다음 심장 기능 상실, 암 기질의 형성을 포함하여 임상 기관의 스펙트럼을 기초. 그러나, 섬유 아 세포는 크게 인해 고유의 이질성에, 셀의 잘못 특성화 유형 남아있다. 섬유 아 세포의 분리를위한 기존의 방법은 근본적으로 세포의 표현형과 행동에 영향을 미치는 세포 배양의 시간이 필요합니다. 따라서, 섬유 아세포 생물학을 연구 많은 연구는 체외 조작에 의존하고 정확하게 생체 내에서 섬유 아세포의 동작을 캡처하지 않습니다. 이러한 문제를 극복하기 위해, 우리는 preservi함으로써, 세포 배양을 필요로하지 않는 성인 마우스의 등쪽 피부 섬유 아세포의 분리에 대한 FACS 기반 프로토콜을 개발생리적 전사 및 각 셀의 프로테옴 프로파일을 겨. 우리의 전략은 혈통 음의 게이트를 통해이 아닌 중간 엽 계통의 배제를 허용 (린 -)가 아니라 특정 표면 마커를 발현하는 섬유 아 세포의 하위 집단의 사전 선택 또는 농축을 방지하고이 이질적인에서 가능한 한 포함 될 수있는 긍정적 인 선택 전략을보다 세포 유형.

이 방법 (도 1)의 유효 기간은 27 일 최근의 간행물에 상세하게 검사 할 수있는 방법의 다수 확인되었다. 이러한 분류 세포 대량 세포와 갓 분류 세포의 단일 세포 전사 분석의 면역 세포를 포함한다. 섬유 아세포를 정렬 직접보다는 더 정확하게 문화에 의존하는 것은 자신의 생체 표현형을 캡처합니다. 오히려 긍정적 인 선택 방식보다 혈통 부정적인 고갈 방식 (그림 2A)를 ?...

Watch this Scientific Journal Video about Murine Dermal Fibroblast Isolation by FACS at JoVE.com

Murine Dermal Fibroblast Isolation by FACS

FACS에 의해 쥐 피부 섬유 아세포 격리

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

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Developmental Biology

21.0K 조회수.  Stanford University School of Medicine. Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

비디오: Murine Dermal Fibroblast Isolation by FACS

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

등쪽 마우스 피부에서 헤어 난포 줄기 세포와 표피 각질 형성 세포를 분리

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

연구

발생학

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

1 차적인 뮤린 멜라닌세포 및 섬유아세포 배양물의 급속한 생성

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

암 연구학

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished.
Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

This manuscript describes a FACS-based protocol for isolation of papillary and reticular fibroblasts from human skin. It circumvents in vitro culture which was inevitable with the commonly used isolation protocol via explant cultures. The emanating fibroblast subsets are functionally distinct and display differential gene expression and localization within the dermis.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have ...

유전학

FACS-Isolation and Culture of Fibro-Adipogenic Progenitors and Muscle Stem Cells from Unperturbed and Injured Mouse Skeletal Muscle

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along fibrogenic, adipogenic, osteogenic, or chondrogenic lineage. Together with muscle stem cells (MuSCs), FAPs play a critical role in muscle homeostasis, repair, and regeneration, while actively maintaining and remodeling the extracellular matrix (ECM). In pathological conditions, such as chronic damage and muscular dystrophies, FAPs undergo aberrant activation and differentiate into collagen-producing fibroblasts and adipocytes, leading to fibrosis and intramuscular fatty infiltration. Thus, FAPs play a dual role in muscle regeneration, either by sustaining MuSC turnover and promoting tissue repair or contributing to fibrotic scar formation and ectopic fat infiltrates, which compromise the integrity and function of the skeletal muscle tissue. A proper purification of FAPs and MuSCs is a prerequisite for understanding the biological role of these cells in physiological as well as in pathological conditions. Here, we describe a standardized method for the simultaneous isolation of FAPs and MuSCs from limb muscles of adult mice using fluorescence-activated cell sorting (FACS). The protocol describes in detail the mechanical and enzymatic dissociation of mononucleated cells from whole limb muscles and injured tibialis anterior (TA) muscles. FAPs and MuSCs are subsequently isolated using a semi-automated cell sorter to obtain pure cell populations. We additionally describe an optimized method for culturing quiescent and activated FAPs and MuSCs, either alone or in coculture conditions.

The precise identification of fibro-adipogenic progenitor cells (FAPs) and muscle stem cells (MuSCs) is critical to studying their biological function in physiological and pathological conditions. This protocol provides guidelines for the isolation, purification, and culture of FAPs and MuSCs from adult mouse muscles.

교란되지 않고 손상된 마우스 골격근에서 섬유 지방 형성 전구 세포 및 근육 줄기 세포의 FACS 분리 및 배양

Fibro-adipogenic progenitor cells (FAPs) are a population of skeletal muscle-resident mesenchymal stromal cells (MSCs) capable of differentiating along ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

비통합 방법을 사용하여 복부 피부에서 성인 인간 진피 섬유 아세포의 분리 및 유도 만능 줄기 세포의 생성

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

생체공학

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

형광 활성 세포 정렬 (FACS)에 의해 신선한 조직으로부터 정상 및 암 관련 섬유 아세포의 분리

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

생물학

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

초음파 증강 원발성 섬유아세포 분리

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

의학

Isolation, Culture, and Characterization of Primary Dermal Fibroblasts from Human Keloid Tissue

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of the biological function and molecular mechanisms of keloids, as well as new therapeutic strategies for treating them. The traditional method of obtaining primary fibroblasts has limitations, such as poor cellular state, mixing with other types of cells, and susceptibility to contamination. This paper describes an optimized and easily reproducible protocol that could reduce the occurrence of possible issues when obtaining fibroblasts. In this protocol, fibroblasts can be observed 5 days after isolation and reach nearly 80% confluency after 10 days of culture. Then, the fibroblasts are passaged and verified using PDGFR&#945; and vimentin antibodies for immunofluorescence assays and CD90 antibodies for flow cytometry. In conclusion, fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research.

This study describes an optimized protocol for establishing primary fibroblasts from keloid tissues that can effectively and steadily provide pure and viable fibroblasts.

인간 켈로이드 조직에서 일차 진피 섬유아세포의 분리, 배양 및 특성화

Fibroblasts, the major cell type in keloid tissue, play an essential role in the formation and development&#160;of keloids. The isolation and culture of ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

성인 심장 섬유 아세포 및 근섬유 아세포의 격리 및 특성화

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.