이 연구는 국립 과학 재단의 중국 (21474057 및 21604076)에 의해 지원 되었다.

주의: 사용 하기 전에 모든 관련 물질 안전 데이터 시트 (MSDS)를 참조 하십시오. 증기 두건 및 개인 보호 장비 (보호 안경, 보호 장갑, 랩 코트, 등)의 사용을 포함 하 여 화학 실험을 수행할 때 적절 한 안전 관행을 사용 하십시오. 프로토콜 처리 기술 (살 균, 세포 복구, 셀 뿌리고, 세포 동결, 셀 얼룩, 등) 표준 셀 필요.
 1. 준비의 Hydrogels <ol>benzaldehyde의 합성 종료 디 기능성된 폴 리 에틸렌 글리콜 (PEG DF) <ol>사전 건조 목이 <li><li>폴리머 <ol><li>4.00 g PEG (MW 4000, 1.00 mmol), 무게 둥근 바닥 플라스 크 (250 mL)에 그것을 전송 하 고 톨루엔 (100 mL) 추가. 
			참고: 다른 분자 무게를 가진 다른 구슬 하이드로 겔 형성을 위해 일할 수 있지만 다른 강성으로 이어질 것입니다.</li>
			<li>열 열 총 (또는 핫 플레이트 약 40 ° C) 약간에 의해 모든 고분자를 분해 하는 데 도움이 솔루션. 모든 고분자 분해 후 모든 용 매를 제거 하는 증발 기를 사용 합니다. 이 분해 과정과 건조을 고분자 말린된 나무 못을 두 번 반복. 
			주의:이 주의 연기 후드에서 수행 되어야 합니다. 톨루엔은 인화성.</li>
		</ol></li> <li>말뚝의 benzaldehyde 종료 반응 <ol><li>플라스 크를 자력 및 tetrahydrofuran (THF, 100 mL)을 추가 하 고는 교 반기를 사용 하 여 말뚝을 분해. 모든 고체를 완전히 해산 솔루션 4-carboxybenzaldehyde (0.90 g, 6.0 mmol)과 4-dimethylaminopyridine (0.07 g, 0.6 m m o l)을 순차적으로 추가.</li>
			<li>추가 N, N &#39;-dicyclohexylcarbodiimide (DCC, 1.25 g, 6.0 mmol)를 솔루션에 추가 건조 튜브 무수 CaCl 2 플라스 크에 가득. 실 온에서 교 반 아래 반응 계속 (~ 20 ° C) 약 12 헤 대 한</li>
		</ol></li> <li>후 반응 과정 <ol><li>반응이 완료 된 후 진공 하 여 솔루션에서 생성 된 백색 고체를 필터링. 솔루션을 아래 흰색 고체 침전을 교 반 찬 diethyl 에테르 (500 mL)에 붓으십시오. 필터에 의해 모든 백색 고체를 수집 하 고 증기 두건에서 고체 건조.</li>
			<li>는 THF에 흰색 고체를 다시 해산, 모든 불 용해성 백색 고체 필터링, 흰색 고체 침전을 신선한 찬 diethyl 에테르에 솔루션을 붓는 다 고 그것을 건조. 2 ~ 3 배,이 과정을 반복 하 고 진공 건조 오븐 (20 ° C, 0.1 mbar) 그들을 완전히 건조에 흰색 고체를 배치. 최종 제품으로 백색 분말을 수집: benzaldehyde 종료 DF 못.</li>
		</ol></li></ol></li> <li>Hydrogels의 준비 <ol><li>튜브 (10 mL) DF 못 (0.11 g, 0.028 mmol, 0.22 g, 0.055 mmol, 0.44 g, 0.110 mmol)의 다른 양을 무게 이온된 수 (5.0 mL), 추가 하 고 소용돌이 또는 자력 교 반기를 사용 하 여 도움말 폴리머 분해.</li>
		<li>글리콜 키토 산 (0.495 g, 10 -3 mmol x 6.18) 튜브 (50 mL), 이온된 수 (15.0 mL)에 녹이 고 몇 분 동안 소용돌이 사용 하 여 균질 키토 산 용액 (3 wt %).</li>
		<li>글리콜 키토 산 용액 (0.2 mL, 3 wt %)와 추가 DF 못 솔루션 (0.2 mL) 순차적으로 튜브 (2.0 mL). 소용돌이 사용 하 여 몇 분에 솔루션 양식 hydrogels 균질 혼합. 다른 기계적인 힘의 hydrogels 수 있도록 표 1의 비율에 따라. 
		참고: 튜브 반전 메서드를 사용 하는 하이드로 겔은 이미 형성 여부 확인.</li>
	</ol></li> <li>유동성 분석 <ol><li>병렬 강판으로 회전 고분자에 유동성 분석 수행 (직경: 20 m m). 겔 화 테스트에 대 한 낮은 접시에 글리콜 키토 산 용액 (0.2 mL, 3 wt %)를 확산. 다음, 키토 산 솔루션 표면에 고르게 dropwise DF 못 수성 솔루션 (0.2 mL, 2 wt %)를 추가 하 고 신속 하 게는 피 펫과 혼합. 낮은 위 격판덮개를 아래로 하 고 테스트 시작</li>
		<li>하이드로 겔에 대 한 &#39; s 강성 테스트를 원형으로 한 히드로 컷 (직경: 20mm) 저장 모듈러스를 측정 (G &#39;) 주파수 분석 1% 변형에 대 한 값. 전형적인 G &#39; 6.3 rad s 값 &#8722; 1 표 1에 나열 됩니다. 
		참고: 동적 본드는 히드로의 안정화를 위해 하이드로 겔 형성 후 하이드로 겔 0.5 h의 유동성 분석 수행.</li>
		<li>는 하이드로 겔에 대 한 &#39; s 자체 healable 속성 테스트 조각 하는 하이드로 겔을 잘라 하 고 필수적인 부분에 자체를 함께 조각 수집. 원을 만들기 위해 하이드로 겔 조각 잘라 (직경: 20mm) 유동성 분석을 수행 하는 고분자에.</li>
	</ol></li></ol> 2. Hydrogels에 3D 세포 배양 <ol><li>히드로 겔 화 솔루션의 준비 <ol><li>세포 배양 (RPMI-1640, 2.0에에서 추가 무게 DF 말뚝 (0.44 g, 0.11 mmol) 무 균 튜브 (4.0 mL) mL), 및 소용돌이 또는 교 반기를 사용 하 여 고분자 폴리머 솔루션 (20 wt %)를 분해 하는 데 도움이. 주사기 (10.0 mL)에서 솔루션을 로드 하 고 다음 미크론 박테리아 못 벗어난 필터 (0.22 μ m)를 통해 그것을 전달 하 여 소독.</li>
		<li>는 글리콜 키토 산 (0.165 g, 10 -3 mmol x 2.06) 무 균 튜브 (15.0 mL)에 무게, 셀 문화 미디어 (RPMI-1640, 4.0 mL), 그리고 글리콜 키토 산 용액 (4.0 wt %)를 폴리머 분해 있도록 소용돌이에 추가. 주사기 (10.0 mL)에서 솔루션을 로드 하 고 0.22 μ m 박테리아 못 벗어난 필터와 필터.</li>
	</ol></li> <li>Hydrogels에서 세포 배양 주의: 수행 모든 세포 조직 문화 후드에서 관련된 절차. 살 균 기술의 기본 지식으로 예상 된다.
	<ol><li>세포 현 탁 액 <ol><li>RPMI-1640 년에 문화 L929 세포의 준비 중간 보충 10 %FBS, 5% 페니실린 (10 mL)는 페 트리에서 접시 (직경 10 cm), 및 37 ° C, 5% CO 2에서 품 어. 매체를 사용 하기 전에 매일 변경.</li>
			<li>Trypsin (0.025 w/v %)과 EDTA를 포함 하는 PBS를 가진 L929 세포를 수확 (0.01 w/v %), 다음 원심 (70 x g, 5 분) 하 고 다시 RPMI 1640 매체 (1.0 mL)에 셀을 일시 중지. 셀 피 세 보드의 표준 동작을 사용 하 여 계산을 수행 합니다. 다시 셀을 셀 농도 조정 중단 ~ 3.75 × 10 6 셀/mL.</li>
		</ol></li> <li>Hydrogels에 셀 캡슐화 <ol><li>L929 세포 정지 (0.4 mL, 10 6 세포 mL -1 x 3.75) 글리콜 키토 산 용액 (0.4 mL) 튜브 (4.0 mL) 소용돌이 의해 혼합. 공초점 레이저 페 트리 접시의 중앙에 L929/글리콜 키토 산 용액 (0.8 mL)를 플라스틱 (직경 2.0 cm). DF 못 솔루션 (0.2 mL) 같은 그릇에 플라스틱 하 고 부드럽게 솔루션을 조합 하 여 하이드로 겔 형성 유도 플라스틱. 
			참고: 페 트리 접시를 기울임으로써 하이드로 겔 형성 평가.</li>
			<li>직접 세포 배양에 대 한 추가 히드로 위에 RPMI 1640 문화 미디어 (1.0 mL)의 추가 금액. 인큐베이터 (37 ° C, 5% CO 2)에서 포함 하는 셀 hydrogels (1.0 mL, 1.5 wt % 글리콜 키토 산, 4.0 wt %DF 못, 1.5 × 10 6 세포 mL -1)을 넣고 매일 매체를 변경. 하루 1, 3, 5, 및 7 셀 캡슐화 후에 셀 이미징에 대 한 준비.</li>
		</ol></li> <li>Hydrogels 주입 후에 3 차원 셀 후 문화, 준비 로드 셀 하이드로 겔 (1.0 mL 단계 2.2.2 참조) 주사기 (10.0 mL, 48 G 바늘)에. 하이드로 겔 형태의 후 천천히 confocal 영상에 대 한 페 트리 접시에는 히드로 주입. 위에 하이드로 겔 문화 미디어 (1.0 mL)의 추가 금액을 추가 하 고 매일 그것을 변경할. (37 ° C, 5% CO 2) 인큐베이터에서 배양 접시를 넣고 나중 이미징에 대 한 준비. 
		주의: 검사 하 고 주사기의 안전 작업 프로토콜 따라.</li>
	</ol></li> <li>세포 생존 능력 분석 <ol><li>Confocal 관찰 <ol>
			<li>2 번 PBS (1.0 mL)와 hydrogels 린스. Fluorescein diacetate (FDA, 0.5 mL, 0.05 mg/mL)와 15 분 동안 propidium 요오드 화물 (PI, 0.5 mL, 0.08 mg/mL) 솔루션 hydrogels 얼룩. 모든 용 매를 제거 하는 얼룩, 후.</li>
			<li>Hydrogels 488의 여기 파장에서 confocal 현미경을 사용 하 여 관찰 및 543 nm 라이브 시각화 및 죽은 세포, 각각. Z-스택 전체 셀의 동등한 배급을 확인 하기 위해 hydrogels의 모든 2 µ m 깊이 통해 받아. 
			참고: FDA 얼룩 라이브 셀 동안 PI 죽은 세포 얼룩.</li>
		</ol></li> <li>튜브 (4.0 mL)로 5 분 및 피 펫에 대 한 초 산 (HAc, 3 v %, 1.0 mL)와 히드로 (1.0 mL)을 저하. 원심 분리기 (70 x g, 5 분)에 의해 세포를 수집 하 고 다시 RPMI 1640 세포 배양 매체 (1.0 mL)에 셀을 일시 중단. 셀 보드를 계산 하는 혈액을 사용 하 여 계산 수행.</li>
	</ol>
	</li>
</ol>

<ol>
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W., Baaijens, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+viscoelastic+behavior+of+subcutaneous+adipose+tissue.">Linear viscoelastic behavior of subcutaneous adipose tissue.</a> Biorheology. 45 (6), 677-688 (2008).</li><li>Banerjee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+hydrogel+modulus+on+the+proliferation+and+differentiation+of+encapsulated+neural+stem+cells.">The influence of hydrogel modulus on the proliferation and differentiation of encapsulated neural stem cells.</a> Biomaterials. 30 (27), 4695-4699 (2009).</li><li>Benoit, D. S., Schwartz, M. P., Durney, A. R., Anseth, K. 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저자는 공개 없다.

하이드로 겔이이 프로토콜 (그림 1)에 두 가지 주요 구성 요소: 자연 폴리머 글리콜 키토 산 합성 benzaldehyde 종료 폴리머 gelator DF 못, 두 생체 재료입니다. DF 말뚝의 합성 단계 수정 반응을 사용 하 여 제공 됩니다. 분자량 4000의 PEG 하이드로 겔 강성 뿐만 아니라 가용성, 수정 효율성의 문제에서이 프로토콜에 선정 되었다. 다른 기계적인 힘을 가진 hydrogels의 시리즈는 다른 고체...

Hydrogels는 많은 양의 물과 부드러운 기계적 성질, 가교 된 고분자 재료 그리고 그들은 많은 바이오-의료1,2에 사용 되었습니다. Hydrogels는 vivo에서세포에 대 한 생리 적인 환경에 매우 비슷한 부드럽고 젖은 환경을 제공할 수 있습니다. 따라서, hydrogels 3D 셀 문화3,4건설 하는 가장 인기 있는 기계 중 하나가 되고있다. 2D 페 트리 접시 세포 배양에 비해, 3 차원 세포 배양 한 세포 외 기질 (ECM) 유사 문의 확산 및 감 별 법 목적5조립 하는 셀에 대 한 microenvironment 제공 하 신속 하 게 전진 했다. 또한, hydrogels 천연 고분자를 포함 하 수 증식 및 분화3셀 바이오 호환 및 추진 환경을 제공 하 고 있다. 합성 중합체에서 파생 된 Hydrogels 동물 유래 단백질 이나 바이러스 같은 복잡 한 영향을 제외 그들의 간단 하 고 명확한 구성 선호 됩니다. 3D 세포 배양에 대 한 모든 히드로 후보자, 중 hydrogels 쉽게 준비 하 고 일관 된 속성을 항상 선호 됩니다. 하이드로 겔의 다른 연구 요구 사항에 맞게 속성 조정 시설 뿐만 아니라6으로 중요 하다.
여기 우리는 글리콜 키토 산 기반의 히드로 동적 것 화학, 3D 셀 문화7다목적 히드로 플랫폼을 사용 하 여 손쉬운 준비 소개. 이 방법에서는, 잘 알려진 바이오 호환 글리콜 키토 산 하이드로 겔의 네트워크의 프레임을 설정 하는 데 사용 됩니다. 아미노 그룹은 hydrogels8의 crosslinkages로 동적 것 유대를 형성 하는 폴리머 gelator로 종료 benzaldehyde 폴 리 에틸렌 글리콜과 반응. 동적 것 유대 형성 하 고 기계적으로 조정 가능한 가교 된 네트워크9,,1011hydrogels 부여 주변 역 고 responsively 분해 수 있습니다. 높은 물 내용을, 바이오 호환 재료, 및 조정 가능한 기계적인 힘는 하이드로 겔 3D 셀 문화12,13L929 세포에 대 한 발판으로 성공적으로 적용 됩니다. 여기에 프로토콜 등 폴리머 gelator 합성, 히드로 준비, 셀 포함, 3D 세포 배양 절차를 자세히 설명 합니다.
히드로 동적 것 crosslinkages, 다중 응답 다양 한 바이오-자극 (산/pH, 비타민 B6 파생 pyridoxal, 단백질 papain, 등)을 포함 하 여, 히드로 것을 나타내는 때문에 여러 가지 다른 기능을 보여 줍니다. 8생리 적 조건 분해를 유도 한다. 하이드로 겔은 또한 자체 healable 고 주 사용는 히드로 최소한의 침략 적 주입 방법을 통해 관리 될 수 및 약물 및 세포 배달14,15에서 얻을. 추가 하 여 기능 첨가제 또는 특정 미리 디자인 된 폴리머 gelators는 히드로 얻고 자기, 온도, pH, 등16,17, 이행할 수 있는 같은 특정 속성에 호환 되는 요구 사항 연구의 넓은 범위. 이러한 속성 주 사용 하는 하이드로 겔의 잠재 능력 약물과 생체 외에서 그리고 vivo에서 생물 의학 연구 및 응용 프로그램에서 셀에 대 한 여러 사업자 공개.

<table border="0" cellpadding="0" cellspacing="0" width="1151">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Glycol chitosan</td>
			<td style="width:360px;">Wako Pure Chemical Industries</td>
			<td style="width:131px;">39280-86-9</td>
			<td style="width:385px;">90% degree of deacetylation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Carboxybenzaldehyde</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>619-66-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N, N&#39;-dicyclohexylcarbodiimide</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>538-75-0</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride anhydrous</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>10043-52-4</td>
			<td>96%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-dimethylamiopryidine</td>
			<td>Shanghai Aladdin&#160;Bio-Chem Technology Co.,LTD</td>
			<td>1122-58</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polyethyleneglycol</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>5254-43-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetrahydrofuran</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>109-99-9</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Toluene</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>108-88-3</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethyl ether</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>60-29-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>64-19-7</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anhydrous CaCl2</td>
			<td>Sino-pharm Chemical Reagent</td>
			<td>10043-52-4</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>596-09-8</td>
			<td>99%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium iodide&#160;</td>
			<td>Sigma</td>
			<td>25535-16-4</td>
			<td>94%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI-1640 culture media</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA</td>
			<td>Gibco</td>
			<td></td>
			<td>0.25%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Solarbio</td>
			<td></td>
			<td>0.01 M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin streptomycin solution</td>
			<td>Hyclone</td>
			<td></td>
			<td>10,000 U/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rheometer</td>
			<td>TA Instrument</td>
			<td></td>
			<td>AR-G2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>710-3channel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L929 Cells</td>
			<td>ATCC</td>
			<td></td>
			<td>NCTC clone 929; L cell, L929, derivative of Strain L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Evaporator</td>
			<td>EYELA</td>
			<td></td>
			<td>N-1100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 guage needle</td>
			<td>ShanghaiZhiyu Medical Material Co., LTD</td>
			<td></td>
			<td>48-guage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DM3000 B</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope software</td>
			<td>Imaris</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat gun</td>
			<td>Confu</td>
			<td></td>
			<td>KF-5843&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish</td>
			<td>NEST</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of chitosan-based injectable hydrogels and its application in 3d cell culture

프로토콜 키토 산 기반 hydrogels 동적 것 화학을 사용 하 여 준비 하기 손쉬운 효율적이 고 다양 한 메서드를 제공 합니다. 하이드로 겔 합성된 benzaldehyde 종료 폴리머 gelator와 글리콜 키토 산의 솔루션을 혼합 하 여 준비 되 고 hydrogels 실 온에서 몇 분에 효율적으로 얻을 수 있습니다. 글리콜 키토 산, 폴리머 gelator, 및 물 내용 사이 다양 한 비율에 의해 다른 겔 화 시간 및 강성 다목적 hydrogels 얻을 수 있습니다. 손상, 그것의 외관 및 계수, crosslinkages로 동적 것 유대의 가역 때문에 히드로 복구할 수 있습니다. 이 자체 healable 속성에는 히드로 주입 과정 후 필수 대량 하이드로 겔을 압착된 조각에서 자체 치유 될 수 있기 때문에 주사 될 수 있습니다. 하이드로 겔 멀티 동적 것 유대의 다른 평형 상태 때문에 많은 바이오-액티브 자극에 반응 이기도합니다. 이 히드로 바이오 호환으로 확인 됐다 고 L929 마우스 fibroblast 세포 포함 된 다음과 같은 표준 절차 이었고 셀 확산 쉽게 3D 세포 배양 과정에 의해 평가 되었다. 히드로 셀에 대 한 3D 환경의 생리 적 모방 이익이 되었다 다른 연구에 대 한 조정 플랫폼을 제공할 수 있습니다. 다중 응답, 자체 healable 및 주 사용 속성을 함께 hydrogels 마약과 셀 미래 바이오-의료 응용 프로그램에 대 한 여러 사업자로 잠재적으로 적용할 수 있습니다.

하이드로 겔 준비 및 3D 세포 배양으로 그것의 사용에이 프로토콜의 프레 젠 테이 션이 회로도 그림 1에 제공 됩니다. 하이드로 겔의 내용과 다른 기계적인 힘으로 준비 하는 비율의 정보는 표 1에 요약 됩니다. 히드로 자체 healable 고 유동성 재산 저장 모듈러스 주파수 테스트 그림2에서 대 여 히드로 강성을 선물 한다. 셀 공초점 영상과 hydro...

Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

여기는 키토 산 기반의 주 사용 hydrogels 동적 것 화학을 사용 하 여 손쉬운 준비에 설명 합니다. 하이드로 겔의 기계적 강도 및 3D 세포 배양에서의 응용 프로그램을 조정 하는 방법은 제시 하는.

주 사용 Hydrogels 키토 산 기반 및 3D 세포 배양에서의 응용의 준비

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

preparation-chitosan-based-injectable-hydrogels-its-application-3d

Research

JoVE Journal

Chemistry

18.9K 조회수.  Tsinghua University. 여기는 키토 산 기반의 주 사용 hydrogels 동적 것 화학을 사용 하 여 손쉬운 준비에 설명 합니다. 하이드로 겔의 기계적 강도 및 3D 세포 배양에서의 응용 프로그램을 조정 하는 방법은 제시 하는.

비디오: Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

합성, 세포 전달 및<em&gt; 생체 내</em&gt; 덴드리머 기반 pH 센서의 응용 프로그램

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

연구

화학

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

초성능 액체 크로마토그래피에 의한 조성 분석을 위한 세균세포벽의 격리 및 준비

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures in multi-gram scale quantities. The goal of this manuscript is to clearly detail how to handle and prepare these unique carbenes such that a synthetic chemist of any skill level can work with them. The two carbenes described are a diamidocarbene (DAC, carbene 1) and a monoamidoaminocarbene (MAAC 2). These carbenes are highly electron-deficient and as such display reactivity profiles that are atypical of more traditional N-heterocyclic carbenes. Additionally, these two carbenes only differ in their electrophilic character and not their steric parameters, making them ideal for studying how carbene electronics influence reactivity. To demonstrate this phenomenon, we are also describing the activation of white phosphorus (P4) using these carbenes. Depending on the carbene used, two very different phosphorus-containing compounds can be isolated. When the DAC 1 is used, a tris(phosphaalkenyl)phosphane can be isolated as the exclusive product. Remarkably however, when MAAC 2 is added to P4 under identical reaction conditions, an unexpected carbene-supported P8&#160;allotrope of phosphorus is isolated exclusively. Mechanistic studies demonstrate that this carbene-supported P8allotrope forms via a [2+2] cycloaddition dimerization of a transient diphosphene which has been trapped by treatment with 2,3-dimethyl-1,3-butadiene.

Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

화이트 인의 활성화에 카르 보닐 장식 카르 벤의 제조 및 사용

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

EOB-DTPA와의 조지아 (III) 단지에 대한 조사 그것<sup&gt; (68)</sup&gt; 조지아 방사성 표지 된 아날로그

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We present methods for depositing and passively manipulating C60 molecules on rippled graphene that advance the pursuit of realizing applications involving 1D C60/graphene hybrid structures. The techniques applied in this exposition are geared towards high vacuum systems with preparation areas capable of supporting molecular deposition as well as thermal annealing of the samples. We focus on C60 deposition at low pressure using a homemade Knudsen cell connected to a scanning tunneling microscopy (STM) system. The number of molecules deposited is regulated by controlling the temperature of the Knudsen cell and the deposition time. One-dimensional (1D) C60 chain structures with widths of two to three molecules can be prepared via tuning of the experimental conditions. The surface mobility of the C60 molecules increases with annealing temperature allowing them to move within the periodic potential of the rippled graphene. Using this mechanism, it is possible to control the transition of 1D C60 chain structures to a hexagonal close packed quasi-1D stripe structure.

Preparation and Characterization of C60/Graphene Hybrid Nanostructures

Here we present a protocol for the fabrication of C60/graphene hybrid nanostructures by physical thermal evaporation. Particularly, the proper manipulation of deposition and annealing conditions allow the control over the creation of 1D and quasi 1D C60 structures on rippled graphene.

준비 및 C60/Graphene 하이브리드 Nanostructures의 특성화

Physical thermal deposition in a high vacuum environment is a clean and controllable method for fabricating novel molecular nanostructures on graphene. We ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

Nanodiamonds 및은 나노 입자의 그것의 감소의 바이오 영감 Polydopamine 표면 수정

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Photogeneration of N-Heterocyclic Carbenes: Application in Photoinduced Ring-Opening Metathesis Polymerization

We report a method to generate the N-heterocyclic carbene (NHC) 1,3-dimesitylimidazol-2-ylidene (IMes) under UV-irradiation at 365 nm to characterize IMes and determine the corresponding photochemical mechanism. Then, we describe a protocol to perform ring-opening metathesis polymerization (ROMP) in solution and in miniemulsion using this NHC-photogenerating system. To photogenerate IMes, a system comprising 2-isopropylthioxanthone (ITX) as the sensitizer and 1,3-dimesitylimidazolium tetraphenylborate (IMesH+BPh4-) as the protected form of NHC is employed. IMesH+BPh4- can be obtained in a single step by anion exchange between 1,3-dimesitylimidazolium chloride and sodium tetraphenylborate. A real-time steady-state photolysis setup is described, which hints that the photochemical reaction proceeds in two consecutive steps: 1) ITX triplet is photo-reduced by the borate anion and 2) subsequent proton transfer takes place from the imidazolium cation to produce the expected NHC IMes. Two separate characterization protocols are implemented. Firstly, CS2 is added to the reaction media to evidence the photogeneration of NHC through formation of the IMes-CS2 adduct. Secondly, the amount of NHC released in situ is quantified using acid-base titration. The use of this NHC photo-generating system for the ROMP of norbornene is also discussed. In solution, a photopolymerization experiment is conducted by mixing ITX, IMesH+BPh4-, [RuCl2(p-cymene)]2 and norbornene in CH2Cl2, then irradiating the solution in a UV reactor. In a dispersed medium, a monomer miniemulsion is first formed then irradiated inside an annular reactor to produce a stable poly(norbornene) latex.

We describe a protocol to photogenerate N-heterocyclic carbenes (NHCs) by UV irradiation of a 2-isopropylthioxanthone/imidazolium tetraphenylborate salt system. Methods to characterize the photoreleased NHC and elucidate the photochemical mechanism are proposed. The protocols for ring-opening metathesis photopolymerization in solution and miniemulsion illustrate the potential of this 2-component NHC photogenerating system.

N-이종 환 식 Carbenes의 Photogeneration: Photoinduced 반지 개통 Metathesis 중 합에 응용

We report a method to generate the N-heterocyclic carbene (NHC) 1,3-dimesitylimidazol-2-ylidene (IMes) under UV-irradiation at 365 nm to characterize IMes ...

Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, when used together, can indicate the presence of gunshot residue (GSR) by producing a fluorescence signal in the presence of three key analytes (antimony, lead, and organic components of GSR). The protocol describes the synthesis of the biosensors using Escherichia coli (E. coli), and the analytical chemistry methods used to evaluate the selectivity and sensitivity of the sensors. The functioning of the system is demonstrated by using GSR collected from the inside of a spent cartridge casing. Once prepared, the biosensors can be stored until needed and can be used as a test for these key analytes. A positive response from all three analytes provides a presumptive positive test for GSR, while each individual device has applications for detecting the analytes in other samples (e.g., a detector for lead contamination in drinking water). The main limitation of the system is the time required for a positive signal; future work may involve studying different organisms to optimize the response time.

A protocol is presented using synthetic biology techniques to synthesize a set of bacterial biosensors for the analysis of gunshot residue, and to test the functioning of the devices for their intended use using fluorescence spectroscopy.

총 탄 잔류물의 추정 검출을 위한 새로운 세균 바이오 센서의 제조 및 적용

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, ...

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. The versatility of the GSMLCs is demonstrated in the context of a study about etching and growth dynamics of gold nanostructures from a HAuCl4 precursor solution. GSMLCs combine the advantages of conventional silicon- and graphene-based liquid cells by offering reproducible well depths together with facile cell manufacturing and handling of the specimen under investigation. The GSMLCs are fabricated on a single silicon substrate which drastically reduces the complexity of the manufacturing process compared to two-wafer-based liquid cell designs. Here, no bonding or alignment process steps are required. Furthermore, the enclosed liquid volume can be tailored to the respective experimental requirements by simply adjusting the thickness of a silicon nitride layer. This enables a significant reduction of window bulging in the electron microscope vacuum. Finally, a state-of-the-art quantitative evaluation of single particle tracking and dendrite formation in liquid cell experiments using only open source software is presented.

Preparation of Graphene-Supported Microwell Liquid Cells for In Situ Transmission Electron Microscopy

A protocol for preparation of graphene-supported microwell liquid cells for in situ electron microscopy of gold nanocrystals from HAuCl4 precursor solution is presented. Furthermore, an analysis routine is presented to quantify observed etching and growth dynamics.

소이투 투과 전자 현미경 을 위한 그래핀 지원 마이크로웰 액체 세포의 준비

The fabrication and preparation of graphene-supported microwell liquid cells (GSMLCs) for in situ electron microscopy is presented in a stepwise protocol. ...