Name: promoting 3-d aggregation of facs purified thymic epithelial cells with eak 16-ii/eakiih6 self-assembling hydrogel
Uploaded: 2016-06-27T14:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel at JoVE.com

이 작품은 R21 AI113000 (WSM)와 R01 AI123392 (YF)는 국립 보건원 (National Institutes of Health)에 의해 부분적으로 부여 지원되었다.

실험에 사용 된 모든 동물은 앨 건강 네트워크 / 앨 가수 연구소의 기관 동물 관리 및 사용위원회에 의해 검토 및 승인 프로토콜에서 앨-가수 연구소에서 동물 시설에 수용되었다. 1. 콜라게나로 흉선 소화 <ol><li> 수확 흉선 분비. <ol><li> 흉선을 수확 3-5 주령 C57BL6 / J의 이산화탄소에 마우스 (CO 2) 챔버를 안락사. 구체적으로, 3 ~ 5 분 동안 CO 2 유도에서 챔버에 마우스를 배치하고 심장 또는 호흡 정지를 확인하여 죽음을 확인합니다. </li><li> 그 뒷면에있는 시체를 놓고 70 % 에탄올로 몸을 젖은. 피부를 통해 자사의 복부에 날카로운 직선 해부 가위로 작은 수평 절개를합니다. 이 밖에 내부에 켜져 있도록 양쪽 끝 (머리와 다리)에 피부를 당겨. </li><li> 해부 가위 바로 아래에 수평 절단합니다가장 낮은 리브와 옆으로 다이어프램을 잘라. 겸자와 칼 모양의 과정을 잡고 양쪽에 최대 진행 리브의 중간을 잘라. 흉선은 명확하게 볼 수 있도록 갈비 / 흉골을 당깁니다. 참고 : 흉선은 직접 심장의 상단에 놓여 두 엽으로 구성되어 있으며 흰색 반투명​​ 색이다. </li><li> 부드럽게 특종 및 결합 조직 및 세척 용액에 전송 오프 흉선 로브를 당겨, 곡선 집게를 사용하여 (물질의 목록 참조). </li></ol></li><li> 9 ml로 RPMI-1640 (로스웰 파크 메모리얼 연구소 매체-1640) 0.025 ㎎ / ㎖ 정제 된 콜라겐, 10 mM의 HEPES [4- (2- 히드 록시 에틸) -1- 피페 라진 에탄 술폰산, 0.2 ㎎ / ㎖을 혼합하여 소화 용액을 준비 50 ㎖의 원심 분리기 튜브의 DNase I. 사용할 때까지 37 ° C의 물을 욕조에서 소화 용액을 유지합니다. 참고 제조 소화 용액의 양이 하나 5 ㎖ 폴리스틸렌 원형 - 함께 배양 할 수 최대 5 성인 마우스 thymi, 충분바닥 튜브. </li><li> 5-6 ml의 RPMI-1640을 함유 한 60mm의 조직 배양 접시에 흉선 전송. </li><li> 림프구가 유출 할 수있는 작은 조각으로 28 G 인슐린 주사기 바늘 (약 1mm 3 사각형)과 흉선을 해부하다. </li><li> 유리 파스퇴르 피펫으로 배양 접시에 RPMI-1640과 흉선 조각을 씻어. 접시를 기울여과 흉선 조각이 바닥에 정착 할 수 있습니다. 천천히 대기음과 유리 피펫을 사용하여 RPMI-1640 상층 액을 버린다. 이 솔루션은 덜 불투명 때까지 유리 피펫으로 4-5 회 5-6 ml의 RPMI-1640이 세척 단계를 반복합니다. 참고 : 유리 피펫은이 단계에서 권장, 흉선 조각이 흉선 조직의 과도한 손실이 발생, 플라스틱에 집착하는 경향이 있기 때문이다. 절차의 나머지, 플라스틱 피펫을 사용합니다. </li><li> 단계 1.5에 기재된 바와 같이 최종 헹굼 후, 상층 액을 제거한다. 3 ㎖ 소화 솔루션을 추가하고 5 ㎖의 둥근 바닥 페이지로 흉선 조각과 솔루션을 전송olystyrene 튜브. </li><li> 튜브의 회전에 튜브를 넣고 12 rpm의 회전 속도로 6 분 동안 37 ℃에서 배양한다. </li><li> 배양 후, 흉선 단편은 중력에 의해 상기 튜브의 하단에 정착하자. 파스퇴르 피펫으로 상층 액을 수집하고 세척 용액 10 ㎖로 50 ml의 원심 분리기 튜브로 전송할 수 있습니다. 5 분 동안 300 XG에서 원심 분리하고, 상청액을 버리고, 10 mL의 세척 용액으로 세포를 재현 탁. 얼음에 보관하십시오. </li><li> 반복 둥근 바닥 폴리스티렌 튜브 5 ml의에서 흉선 조각에 두 번 1.6-1.8 단계를 반복합니다. 같은 50 ML 튜브에 뜨는 수영장. 주 : 원심 번째 및 세 번째 세척 필요는 없다. </li><li> 50 ㎖의 원심 분리기 튜브에 튜브 및 전송에 남아있는 조각을 무너 뜨리는 최종 소화 한 후, 최대 피펫 2 ml의 플라스틱 혈청 학적 피펫 아래. </li><li> 원심 분리기에서 5 분 동안 300 XG에서 50 ㎖ 튜브 세척 buffe 10ml에 뜨는, 재현 탁 대기음100 μm의 셀 스트레이너를 통해 연구 및 필터. 얼음에 보관하십시오. </li></ol> 불연속 밀도 기울기와 TEC는 2. 심화 <ol><li> 2.4 mL의 밀도 구배 배지 (물 iodixanol의 V / w 60 %) 및 ​​9.6 mL의 세척 용액을 혼합하여 21 % 밀도 구배 용액을 제조 하였다. </li><li> 2.5 ml의 세척 버퍼에 5 분,에 resuspend 300 XG에 단계 1.11에서 50 ㎖ 수집 관에서 해리 흉선 세포를 원심 분리기. </li><li> 세포 현탁액에 20 ml의 RPMI-1640 배지를 추가합니다. </li><li> 전자 피펫으로 10 ㎖의 혈청 학적 피펫에서 21 % 밀도 구배 용액 12 ml를 채 웁니다. 50 mL의 원심 분리 튜브 함유 세포 하단의 혈청 피펫의 선단을 배치하고, 밀도 구배 용액이 중력을 통해 상기 튜브의 하단에 배수 할 수있다. <ol><li> 부드럽게 밀도가 다른 두 층을 생성 완료 피펫에서 밀도 구배 용액을 배출. </li></ol></li><li> 600 XG FO에서 원심 분리기R 스윙 버킷 로터에서 실온에서 20 분. 가장 느린 수준에서 감속 속도를 설정합니다. </li><li> 새로운 50 ㎖ 튜브에 상단 층과의 계면을 전송. 참고 : 열팽창 계수의 차이의 대부분은 인터페이스에 유지됩니다. 림프구는 원심 분리 후 튜브의 바닥에 침전됩니다. 이들 세포는 다른 용도로 사용되는 경우, 3 회 20 ㎖ 세척 용액 펠렛을 헹군다. 세척 완충액 10ml에 세포를 재현 탁. </li><li> 4 ° C에서 6 분 동안 400 XG에 단계 2.6 및 원심 분리기 튜브에 40 ml의 세척 솔루션을 추가 할 수 있습니다. 피펫으로 상층 액, 흡인를 제거합니다. </li><li> 단계 2.7에 기재된 바와 같이 세척하고, 흡인 2 회 더 반복한다. </li><li> 세척 용액 2 ㎖에 재현 탁하고는 혈구 세포 계산. </li></ol> FACS 3. 분리의 열팽창 계수의 차이 <ol><li> 세정액 및 전송로 1 × 106 세포 / 100 ㎕의 농도로 290 단계에서 세포를 조절(유동 분류기의 지침에서 권장하는 폴리 프로필렌 또는 폴리스티렌) 5 ㎖의 둥근 바닥 튜브에 세포. </li><li> 1 × 6 셀 당 2 μL 방지 Fc 수용체 IgG를 추가하고 10 분 동안 4 ° C에서 품어. </li><li> (150 희석, 샘플 당 10 μl의 1) 및 안티는 EpCAM 항 CD45 추가 (1 : 100 희석, 샘플 당 10 μL) 항체 이상 20 분 동안 4 ° C에서 품어. </li><li> 4 ° C에서 6 분 동안 400 XG에 2 ml의 세척 용액을 원심 분리로 세포를 씻으십시오. 피펫으로 뜨는을 대기음. </li><li> 1 ㎖의 1X 인산 완충 생리 식염수 (PBS)으로 세포를 재현 탁. </li><li> 100 μm의 스트레이너를 통해 필터링합니다. </li><li> 정렬 악기의 셀을 적용합니다. 사이드 산란광 (SSC) / 전방 산란 광 (FSC) 패널 (죽은 세포), (13)을 정렬 CD45-는 EpCAM +의 선택 인구에 다음 게이트에서 가장 작은 세포를 제외한 림프구와 TEC 인구를 선택합니다. </li></ol> 4. GTEC / EAK 집계의 eneration 참고 : 시약 준비 및 층류 후드 혼합 셀을 포함하는 단계를 수행합니다. <ol><li> 멸균 된 1.5 ml의 마이크로 원심 튜브에 안티 그의 태그 IgG를 4 μL, 안티는 EpCAM IgG의 8 μL 및 PA / G 2.6 μL를 혼합하여 PA / G 어댑터 착물을 준비하고, 5 분 동안 RT에서 배양한다. </li><li> 세척 용액 및 피펫 낮은 증발, 96 웰 둥근 바닥 조직 배양 플레이트 잘 하나에 100 μL와 5 × 4 세포 / ml로 단계 3.7에서 분류 열팽창 계수의 차이를 조정합니다. 세포에 14.6 μl의 PA / G 어댑터 단지를 추가합니다. 4 ℃에서 20 분 동안 흔들 플랫폼에서 접시를 놓습니다. </li><li> 200 ㎕를 세척 용액으로 한 번 씻으십시오. 6 분 동안 400 XG에 접시를 원심 분리기. 마이크로 피펫으로 상층 액을 버린다. </li><li> 200 ㎕의 10 % 자당 용액으로 한 번 씻으십시오. 6 분 동안 400 XG에 원심 분리기. 마이크로 피펫으로 상층 액을 버린다. </li><li> 30 μl의 10 %의에서 재현 탁 세포물론 당 terile 자당 솔루션입니다. </li><li> EAKIIH6 10 μL (7.5 ㎎ / ㎖)를 첨가하고, 실온에서 5 분 동안 품어. </li><li> 가 TEC 혼합물에 EAK16-II의 20 μL (10 ㎎ / ㎖)를 추가합니다. </li><li> 겔화을 시작하는 시스템에 완전 배지 100 μl를 추가합니다. 실온에서 15 ~ 20 분 동안 플랫폼 통에 접시를 놓습니다. </li><li> 37 ° C에서 인큐베이터에있는 접시를 놓고 5 % CO 2. 2-4 시간 후, 전체 매체의 다른 100 μl를 추가합니다. </li><li> 사용할 때까지 매일 매체를 변경합니다. <ol><li> 대기음 마이크로 피펫을 사용하여 조심스럽게 웰의 벽에 피펫 팁을 배치하여 예열 배지 100 ㎕를 추가 겔을주지 않고 표면으로부터 배지 100 ㎕. </li></ol></li></ol> 가 TEC / EAK 집계 5. 특성 <ol><li> 생체 내에서 TEC / EAK 집계의 기능을 조사하기 위해, 무 흉선 누드의 신장 캡슐에서 O / N 문화와 이식 후 TEC / EAK 하이드로 겔을 수집마우스, 이전 절차 11,14 설명 사용. </li><li> 4~6주 안티 CD4의 칵테일 이식 얼룩을 게시 누드 마우스 -CD8, -CD45에서 EDTA를 함유 혈관으로 50 ~ 100 ㎕의 혈액 샘플을 채취하여 주변의 T 세포의 발달을 모니터링 및 -CD3 (12)는 항체. </li><li> 단일 세포 분석 소프트웨어 (12)를 사용하여 유동 세포 계측법으로 말초 혈액 백혈구 (FCM)에 T 림프구의 백분율을 분석한다. </li></ol>

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Authors declare no conflict of interest.

열팽창 계수의 차이가 흉선 기질의 주된 인구이며 흉선 샘의 구조와 기능에 필수적인 역할을하지만, 이들은 총 흉선 세포 수의 약 0.1 ~ 0.5 %를 차지한다. 그들은 또한 세포 사멸의 높은 비율이 때때로 즉, 치료는 세포 외 기질 (ECM)의 열팽창 계수의 차이를 해리, 콜라게나 제 소화 다음 관찰 깨지기 세포이다. 그들의 희귀 (~ 20 만 마우스의 흉선 당)과 취약성은 그것을 열팽창 계수의 차이를 분...

흉선이 획득 면역계의 기능에 필수적인 병원체 반응성 자기 저항성 T 세포의 다양한 집단의 생성을 담당 차 림프 기관이다. 그것은 흉선 기질의 스폰지 상 3 차원 (3-D) 매트릭스를 통해 이주 현상 흉선 세포는 림프구 전구 세포와 골수 이민 동적 인 기관이다 계통 명세서 및 분화를 거쳐, 최종적으로 이주 성숙한 T 세포. 이 아니라 프로그래밍 프로세스의 성공은 주로 이주 흉선 및 신청 흉선 상피 세포 (TEC는) 설정 및 흉선 미세 환경의 무결성을 유지하기위한 필수적인 가슴샘 기질의 주된 집단 간의 크로스 토크에 따라 다르다. 그 해부학 적 위치 및 고유 함수에 기초하여, TEC는 두 개의 서브 세트들로 분할 될 수있다 : 피질의 열팽창 계수의 차이이다 respon (cTECs)자기 MHC (주요 조직 적합성 복합체)을 선택하기위한 sible은 T 세포 (긍정적 인 선택) 및 자기 반응성 T 세포 (음 선택) 1, 2를 제거하기위한 필수적인 수질의 열팽창 계수의 차이 (mTECs)을 제한. 많은 요인 (예를 들어, 노화, 감염, 방사선 조사, 약물 치료) 손상된 적응 면역의 결과로, 흉선 상피 세포에 돌이킬 수없는 손상을 일으킬 수 있습니다. 수많은 시도에도 불구하고, 흉선 기능을 복원하기 때문에 흉선 미세 환경을 재현하는 어려움에 도전하고있다. 2 차원 환경에서 배양 TEC는 빠르게 thymopoiesis 3,4에 중요한 유전자의 발현이 감소하는 반면 특히 흉선 3 차원 (3-D)의 구성은, 열팽창 계수의 차이의 생존과 기능에 중요하다. EAK16-II (AEAEKAKAEAEAKAK)과 C 말단 histidinylated 아날로그 EAKIIH6 (AEAEKAKAEAEAKAKHHHHHH)은 저 분자량, 탈 물 공급에 가용성 인 양친 매성 올리고 펩타이드이다20 mM의 염화나트륨 (인간 체액 정상 염 농도 154 mM)을보다 높은 이온 강도에 노출되었을 때 R, β-브릴을 형성 겔화하지만 겪는다. 이 환경에 응답 특성은 3 차원 구조를 형성하기 위해 그들에게 다목적 빌딩 블록을 만든다. 단지 단백질 약물과 다른 생체 분자를 고정 할 수있는 어댑터 역할을 할 수 있습니다 (: EAKII-H6에 그의 태그는 도킹 메커니즘을하는 항 그의 IgG를 / 된 Fc 결합 재조합 단백질 A / G (PA / G는 αH6) 제공 예를 들어 하이드로 겔 합성 5-8 항체). 우리는 이전에 하이드로 겔에 고정 형광 표지의 IgG 분자 최대 십삼일 9 주사 부위에 유지 될 수 있음을 증명하고있다. αH6 때 또한 : 안티 - CD4 IgG를 함께 고정 PA / G 어댑터가 EAK16-II / EAKIIH6 (EAK) 하이드로 겔에 첨가하고, CD4 + T 세포는 특별히 (10)를 체포했다. 유사한 기술을 사용하여, 최근 입증하는 TEC는 (C)의 3-D 통합(: PA / G : αEpCAM αH6) TEC-특정 안티는 EpCAM 항체를 운반하는 어댑터 단지와 EAK 하이드로 겔에 승진 울드. 누드 마우스의 신장 캡슐 밑에 이식하면 EAK 히드로 겔에 포함 된 TEC 클러스터 효과적으로 생체 내에서 기능적 11,12 T 세포의 발달을 지원할 수있다. 여기서 우리는 신속하고 효과적으로 형광 - 활성화 된 세포 (FACS)를 정렬과 열팽창 계수의 차이를 정화하고 EAK 하이드로 젤 시스템과 3 차원 TEC 응집체를 생성하기 위해 방법을 도시한다.

<table><tbody><tr><td>&lt;strong&gt;TEC Isolation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>70% Ethanol</td><td>Decon Laboratories</td><td>2701(1 Gallon)</td><td>Ethanol 200 Proof, deionized water</td></tr><tr><td>Dissecting scissors, straight</td><td>Fine Science Tools, Inc.</td><td>91460-11</td><td></td></tr><tr><td>Graefe forceps, straight</td><td>Fine Science Tools, Inc.</td><td>11053-10</td><td></td></tr><tr><td>Graefe forceps, curved</td><td>Fine Science Tools, Inc.</td><td>11052-10</td><td></td></tr><tr><td>Washing solution</td><td></td><td></td><td>1x PBS, 0.1% BSA, 2&amp;nbsp;mM EDTA</td></tr><tr><td>1x PBS (Phosphate buffered saline)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>BSA (Bovine serum albumin)</td><td>Sigma</td><td>A1470-100G</td><td></td></tr><tr><td>EDTA (ethylenediaminetetraacetic acid)</td><td>Invitrogen</td><td>15575-038</td><td></td></tr><tr><td>Digestion solution</td><td></td><td></td><td>9 ml RPMI-1640, 0.025&amp;nbsp;mg/ml Liberase TM Research grade, 10&amp;nbsp;mM HEPES, 0.2&amp;nbsp;mg/ml DNaseI</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11879-020</td><td></td></tr><tr><td>Liberase TM Research Grade</td><td>Roche</td><td>05 401 127 001</td><td>referred as &quot;purified collagenase&quot;</td></tr><tr><td>1&amp;nbsp;M HEPES</td><td>Lonza</td><td>17-737E</td><td></td></tr><tr><td>Dnase I</td><td>Roche</td><td>10 104 159 001</td><td></td></tr><tr><td>50&amp;nbsp;ml Centrifuge tube</td><td>Corning</td><td>430290</td><td></td></tr><tr><td>60&amp;nbsp;mm tissue culture dish</td><td>Falcon</td><td>353002</td><td></td></tr><tr><td>1/2&amp;nbsp;cc U-100 Insulin syringe 28&amp;nbsp;1/2&amp;nbsp;G</td><td>Becton Dickinson</td><td>329461</td><td></td></tr><tr><td>5&amp;nbsp;ml Polystyrene round-bottom tube</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>5&amp;nbsp;ml glass pipet</td><td>Fisher Healthcare</td><td>13-678-27E</td><td>Use for rinsing the thymic fragments. Thymic fragments tend to stick to the wall with plastic pipets.</td></tr><tr><td>MACSmix tube rotator</td><td>Miltenyi</td><td>130-090-753</td><td></td></tr><tr><td>100&amp;nbsp;&amp;micro;m Cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>Density gradient medium: OptiPrep</td><td>Axis-Shield</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cell Sorting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>5&amp;nbsp;ml Polypropylene round-bottom tube</td><td>Falcon</td><td>352063</td><td></td></tr><tr><td>Anti-mouse CD16/CD32 (Fc Block)</td><td>BD Biosciences</td><td>553142</td><td>Use as undiluted, 2&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>Anti-mouse CD45-PercpCy5.5</td><td>eBioscience</td><td>45-0451-80</td><td>Use at 1:150, 10&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>Anti-mouse CD326 (EpCAM)-PE</td><td>eBioscience</td><td>12-5791-82</td><td>Use at 1:100, 10&amp;nbsp;&amp;micro;l per sample</td></tr><tr><td>BD Influx</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Single cell analysis software</td><td>FlowJo</td><td></td><td></td></tr><tr><td>&lt;strong&gt;EAK Gel Assembly&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-His-Tag</td><td>AnaSpec</td><td>29673</td><td>&quot;anti-His-Tag IgG&quot;</td></tr><tr><td>Purified anti-mouse CD326 (EpCAM)</td><td>BioLegend</td><td>118202</td><td>&quot;anti-EpCAM IgG&quot;</td></tr><tr><td>Recombinant protein A/G</td><td>Pierce Biotechnology</td><td></td><td></td></tr><tr><td>1.5&amp;nbsp;ml Safe-Lock Tubes, Biopur, Sterile</td><td>Fisher Healthcare</td><td>05-402-24B</td><td>referred as &quot;1.5&amp;nbsp;ml microcentrifuge tube&quot;</td></tr><tr><td>96-well, Tissue culture plate, Round-bottom with low evaporation lid</td><td>BD Falcon</td><td>353917</td><td></td></tr><tr><td>Rocking platform: Nutator Mixer no.1105</td><td>BD Clay Adams</td><td></td><td></td></tr><tr><td>10% sucrose</td><td>Sigma</td><td>S0389</td><td>Prepare with sterile distilled water</td></tr><tr><td>EAK16-II (AcNH-AEAEAKAKAEAEAKAK-CONH&lt;sub&gt;2&lt;/sub&gt;)</td><td>American Peptide Company&amp;nbsp;</td><td></td><td>custom synthesized, 10&amp;nbsp;mg/ml</td></tr><tr><td>EAKIIH6 (AcNH-AEAEAKAKAEAEAKAKHHHHHH-CONH&lt;sub&gt;2&lt;/sub&gt;)</td><td>American Peptide Company&amp;nbsp;</td><td></td><td>custom synthesized, 7.5&amp;nbsp;mg/ml</td></tr><tr><td>Complete medium</td><td></td><td></td><td>RPMI-1640, 10% FBS, 1% Pen/Strep, 1% L-glutamine, 1% NEAA, 5&amp;nbsp;mM HEPES, 50&amp;nbsp;&amp;micro;M 2-Mercaptoethanol</td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11879-020</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Atlanta Biologicals</td><td>S11150</td><td>Heat inactivated before use</td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>L-glutamine 200&amp;nbsp;mM (100x)</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>NEAA (non-essential amino acid) 100x</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>1&amp;nbsp;M HEPES</td><td>BioWhittaker</td><td>17-737E</td><td></td></tr><tr><td>2-Mercaptoethanol (100x)</td><td>Millipore</td><td>ES-007-E</td><td></td></tr><tr><td>Platform shaker: The Belly Dancer</td><td>Stovall Life Sciences Inc.</td><td>model: USBDbo</td><td></td></tr></tbody></table>

promoting 3-d aggregation of facs purified thymic epithelial cells with eak 16-ii/eakiih6 self-assembling hydrogel

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

CD45- 간질 세포를 풍부하게하는 밀도 구배 분리 프로토콜을 사용의 효과를 조사하기 위해, 인터페이스 석출 림프구 펠렛 모두에서 수확 한 세포를 항 CD45, 항는 EpCAM 항체로 염색 하였다. 두 방지 Ulex Europaeus 응집소 1 (UEA1) 및 안티 MHC 클래스 II 항체는 더 CTEC 및 mTEC 하위 집합을 식별하기 위해 염색 칵테일에 포함되었다. 도 1에 도시 된 바와 같이, 우리는 일상적 ...

Watch this Scientific Journal Video about Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel at JoVE.com

Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

EAK 16-II와 FACS 정화 흉선 상피 세포의 3 차원 집계를 홍보 / EAKIIH6 자기 조립 하이드로 겔을

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing ...

promoting-3-d-aggregation-facs-purified-thymic-epithelial-cells-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.5K 조회수.  Allegheny Health Network. This video demonstrates a protocol to enrich thymic epithelial cells (TECs) with density gradient for FACS isolation. It also shows the use of EAK16-II/EAKIIH6 peptides to promote the TEC aggregate formation. The microenvironments of EAK16-II/EAKIIH6 hydrogel provide the 3-D configuration necessary to maintain the survival and function of the TECs.

비디오: Promoting 3-D Aggregation of FACS Purified Thymic Epithelial Cells with EAK 16-II/EAKIIH6 Self-assembling Hydrogel

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

수질 흉선 상피 세포의 증식, 분화 및 무차별 유전자 발현을 지원하는 3D의 Organotypic 공동 문화 모델

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

연구

발생학

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3 차원 자기 조립 펩티드 히드로겔에서 인간 신경 전구 세포의 배양

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

생체공학

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

단계 성장 PEG-펩타이드 Hydrogels에서 β-세포 Spheroids의 생성 및 복구

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Reaggregate Thymus Cultures

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).

In this video the preparation of 2-dGuo-treated reaggregate thymus cultures is demonstrated.

Thymus 문화 Reaggregate

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and ...

생물학

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

조직 공학을위한 3D Hydrogels에서 셀룰러 봉지

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

캡슐화 및 3d에서 셀의 Immunostaining에 대 한 엘라 스 틴 같은 단백질 Hydrogels의 생산

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

반복 패턴 형성을 위한 하이브리드 젤 큐브 장치에 초기 3D 셀 클러스터 제어

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

<meta charset="utf8"></head><body>iPSC 유래 오가노이드를 이용한 흉선 미세환경 재현

Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.

<meta charset="utf8"></head><body>저자 스포트라이트: 인간 흉선 오가노이드를 활용한 흉선 상피세포 및 T세포 연구의 발전

Here, we describe a protocol for the formation of human iPSC-derived thymus organoids grown in 3D fibrin hydrogels aiming to support thymic epithelial cell (TEC) maturation and prolonged maintenance, as well as thymopoiesis in vitro.

3차원 피브린 하이드로겔에서 인간 흉선 오가노이드의 형성

Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the ...

EAK 16-II와 FACS 정화 흉선 상피 세포의 3 차원 집계를 홍보 / EAKIIH6 자기 조립 하이드로 겔을

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수질 흉선 상피 세포의 증식, 분화 및 무차별 유전자 발현을 지원하는 3D의 Organotypic 공동 문화 모델

3 차원 자기 조립 펩티드 히드로겔에서 인간 신경 전구 세포의 배양

단계 성장 PEG-펩타이드 Hydrogels에서 β-세포 Spheroids의 생성 및 복구

Thymus 문화 Reaggregate

조직 공학을위한 3D Hydrogels에서 셀룰러 봉지

캡슐화 및 3d에서 셀의 Immunostaining에 대 한 엘라 스 틴 같은 단백질 Hydrogels의 생산

반복 패턴 형성을 위한 하이브리드 젤 큐브 장치에 초기 3D 셀 클러스터 제어

3차원 피브린 하이드로겔에서 인간 흉선 오가노이드의 형성

3차원 피브린 하이드로겔에서 인간 흉선 오가노이드의 형성

3차원 피브린 하이드로겔에서 인간 흉선 오가노이드의 형성