이 작품은 연방 교육부 및 연구 (FKZ 0312138A 및 FKZ 316159), EU 구조 기금 (ESF/IVWM-B34-0030/10와 ESF/IVBM-B35-0010/12), 주 Mecklenburg 서쪽 Pomerania 독일과 DFG (DA1296/2-1)에 의해 지원 되었다는 독일 심장 재단 (F/01/12), BMBF (VIP + 00240) 고 젖은 기초. 또한, F.H. 및 오후는 FORUN 프로그램의로 스톡 대학 의료 센터 (889001)에 의해 지원 됩니다.

저자는 충돌의 관심을 선언합니다.

최근 몇 년 동안, CD133+ SCs 등장 유망한 세포 인구로 SC 기반 요법으로 입증 단계 I, II, 및 III에 대 한 임상 시험43,44,,4546, 47 , 48 , 49 , 50 , 51 , ...

CD133+ 이종 줄기 재생 의학에 대 한 잠재력 약속 된 조상 세포 인구를 대표 하는 SCs. 그들의 조 혈, 내 피, 및 조직적 분화 잠재적인1,2,3 수는 CD133+ 세포, 예를 들어, 차별화를 통해 neovascularization 프로세스에 기여 하 으로 새로 형성 선박 및 프로-신생 paracrine 메커니즘4,5,,67에 의해 신호의 활성화.
30 개 이상의 승인 된 임상 시험 (ClinicalTrails.gov)에서 그들의 높은 잠재력에도 불구 하 고 그들의 치료 결과 중인 여전히 논쟁 적인 토론4. 실제로, SCs의 임상 응용 프로그램 낮은 보존 관심과 대규모 초기 세포 죽음5,,89의 기관에 의해 방해 된다. 추가 CD133의 엔지니어링+ SCs 사전 이식 이러한 난제를 극복 도움이 될.
효율적인 세포 치료에 대 한 전제 조건 치료 관련 셀10engraftment를 향상 시키기 위해 대규모 초기 세포 죽음의 감소 될 것 이다. 현재 연구 시연 첫 1-2 h, 이식된 세포 유형의 독립적인 동안 매우 끼얹는다 뇌와 심장 등 장기에 90-99%의 엄청난 셀 손실 또는 응용 프로그램 경로11,12,13 14,15,16,17,18,19,,2021. 관심22,23,,2425,26의 사이트에 대상 셀에 혁신적인 비-침략 적 전략을 가능 하 게 SC 마그네틱 나노 (MNPs)를 사용 하 여 라벨 고 동시에 셀 MRI27 (MPI) 이미징 자성 입자를 사용 하 여 모니터링 하는 것을 허용 한다. 가장 효율적인 vivo에서 정 맥 주입23,,2428 후 셀 지도 보다 로컬 관리 후 사용한 셀 보존 대상 적용 자성된 셀 연구 . 따라서, 우리의 그룹 superparamagnetic 산화 철 나노 입자29의 구성 된 배달 시스템을 설계. 이 기술로, CD133+ SCs와 인간 탯 줄 정 맥 내 피 세포 (HUVECs)는 대상 효율적으로 수, 여 체 외에 시도30,31.
사우스 캐롤라이나 요법에 대 한 또 다른 장애물 초기 세포 죽음32에 기여 하는 이식 후 영향을 받는 조직의 적대적인 염증 성 환경입니다. 여러 사전 컨디셔닝 연구 뿐만 아니라 치료 관련 미르의 응용 프로그램은 테스트33; 그것은 성공적으로 입증 되었습니다 안티 apoptotic 미르 생체 외에서 apoptosis를 억제 고 vivo에서세포 engraftment33을 강화. 이 작은 분자, 20-25의 뉴클레오티드로 구성 메신저 RNAs (mRNAs)의 posttranscriptional 변조기로 서 중요 한 역할을 하 고 따라서 줄기 세포 운명과 행동34를 영향을. 또한, 미르 세 도입 호스트 게놈34에 원치 않는 안정적인 통합을 하지 마십시오.
기본 SCs에 핵 산 (NAs)의 효율적인 도입에 대 한 현재 시도 주로 재조합 바이러스8,35기반으로 합니다. 높은 transfection 효율에도 불구 하 고 재조합 바이러스 조작은 벤치를 머리 맡 번역, 예를 들면, 통제 유전자 발현, pathogenicity, immunogenicity, 및 insertional mutagenesis35에 대 한 주요 장애물이 선물 ,36. 따라서, 폴리머 기반 구조 등 비 바이러스 성 전달 시스템 개발에 중요 하다. 그 중에서 polyethylenimine (페이) 저하, 세포질 통풍 관에서에서 보호 하기 위해 나 결로 등 미르에 대 한 혜택을 제공 하는 유효한 배달 차량 나타내고 endosomal 통해 세포내 릴리스 탈출37,38. 또한, 미르-페이 단지 임상 시험39높은 생체 적합성을 설명 했다. 따라서, 우리의 배달 시스템은 biotinylated의 구성 분기 25 kDa 페이는 streptavidin 입히는 MNP 코어30,,3140에 바인딩된.
이 원고에서는 CD133의 (i)는 수동 격리를 설명 하는 포괄적인 프로토콜 소개+ 사우스 캐롤라이나 사우스 캐롤라이나 제품과의 효율적이 고 부드러운 transfection 전략 (ii)의 상세한 묘사와 인간의 골 수 (BM) 기부에서 한 자석으로 비 바이러스 성 고분자 기반 배달 시스템 CD133의 유전 공학에 대 한+ SCs 미르를 사용 하 여. CD133+ SCs 격리 되며 표면 항 체 기반 자기 활성화 셀 정렬 (맥) 시스템을 사용 하 여 인간의 sternal BM aspirates에서 자석으로 농축. 이후에, 세포 생존 능력 뿐만 아니라 셀 순도 cytometry 사용 하 여 분석 된다. 그 후, 미르/PEI/MNP 단지 준비가 CD133+ SCs는 페. transfection 후 18 h, 통풍 관 효율성과 transfection SC 마커 표현과 세포 생존 능력에의 영향 분석 된다. 또한, transfection 복잡 한 화합물의 세포내 분포의 평가 4 색 레이블 및 구조화 조명 현미경 (SIM)를 사용 하 여 수행 됩니다.

<table>
	<tbody>
		<tr>
			<td>7-AAD</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid with Methylene Blue</td>
			<td>Stemcell Technologies</td>
			<td>7060</td>
			<td>3%</td>
		</tr>
		<tr>
			<td>anti-CD133/2-PE (clone: 293C3)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-090-853</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD34-FITC (clone: AC136)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-081-001</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD45-APC-H7 (clone: 2D1)</td>
			<td>BD Biosciences</td>
			<td>560178</td>
			<td></td>
		</tr>
		<tr>
			<td>rhodamine dye; Atto 565 dye conjugated to biotin</td>
			<td>ATTO-TEC GmbH</td>
			<td>AD 565-71</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS LSRII flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSDiva Software 6.1.2</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich GmbH</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>CD133 antibody-linked superparamagnetic iron dextran particles; CD133 MicroBead Kit</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-049</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase B</td>
			<td>Roche Diagnostics GmbH</td>
			<td>11088831001</td>
			<td></td>
		</tr>
		<tr>
			<td>counting chamber</td>
			<td>Paul Marienfeld GmbH &#38; Co. KG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanine 3 dye labelled precursor miR; Cy3 Dye-Labeled Pre-miR Negative Control #1</td>
			<td>Ambion</td>
			<td>AM17120</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanine 5 dye miR labelling kit; Cy5 dye Label IT miRNA Labeling Kit</td>
			<td>Mirus Bio</td>
			<td>MIR 9650</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Roche Diagnostics GmbH</td>
			<td>10104159001</td>
			<td>(100 U/mL)</td>
		</tr>
		<tr>
			<td>ELYRA PS.1 LSM 780 confocal microscope</td>
			<td>Carl Zeiss Jena GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FcR Blocking Reagent, human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-059-901</td>
			<td></td>
		</tr>
		<tr>
			<td>bright green protein labeling kit; Oregon Green 488 Protein Labeling Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>O10241</td>
			<td></td>
		</tr>
		<tr>
			<td>aqueous mounting medium; Fluoroshield</td>
			<td>Sigma-Aldrich GmbH</td>
			<td>F6182</td>
			<td></td>
		</tr>
		<tr>
			<td>density gradient centrifugation tube; Leukosep Centrifuge Tube</td>
			<td>Greiner Bio-One</td>
			<td>89048-932</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS magnet holder; MACS MultiStand</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS pre-separation filter</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-041-407</td>
			<td>30 &#181;m</td>
		</tr>
		<tr>
			<td>MACS separation column (MS / LS)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-042-201 / 130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS permanent magnet; MACS Separator</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-042-302</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-HV PVDF Filter</td>
			<td>Merck</td>
			<td>SLHV013SL</td>
			<td>0.45 &#956;m</td>
		</tr>
		<tr>
			<td>mouse IgG 2b-PE</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-092-215</td>
			<td></td>
		</tr>
		<tr>
			<td>amine reactive dye; Near-IR LIVE/DEAD Fixable Dead Cell Stain Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>L10119</td>
			<td></td>
		</tr>
		<tr>
			<td>human lymphocyte separating medium; Pancoll</td>
			<td>Pan Biotech GmbH</td>
			<td>P04-60500</td>
			<td>density: 1.077 g/mL</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Pan Biotech GmbH</td>
			<td>P04-53500</td>
			<td>without Ca and Mg</td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Sigma-Aldrich GmbH</td>
			<td>408727</td>
			<td>branched; 25 kDa</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>100 U/mL, 100 &#956;g/mL</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Merck Schuchardt OHG</td>
			<td>1040051000</td>
			<td></td>
		</tr>
		<tr>
			<td>unlabelled precursor miR; Pre-miR miRNA Precursor Negative Control #1</td>
			<td>Ambion</td>
			<td>AM17110</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>eBioscience</td>
			<td>00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse decontamination solution; RNaseZap</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>human lymphocyte medium; Roswell Park Memorial Institute (RPMI) 1640 medium</td>
			<td>Pan Biotech GmbH</td>
			<td>P04-16500</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human cytokine supplement; StemSpan CC100</td>
			<td>Stemcell Technologies</td>
			<td>2690</td>
			<td></td>
		</tr>
		<tr>
			<td>serum-free haematopoietic cell expansion medium; StemSpan H3000</td>
			<td>Stemcell Technologies</td>
			<td>9800</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin MagneSphere Paramagnetic Particles</td>
			<td>Promega Corporation</td>
			<td>Z5481</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma-Aldrich GmbH</td>
			<td>T8154</td>
			<td>0.4 %</td>
		</tr>
		<tr>
			<td>UltraPure EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td>0.5 M; pH 8.0</td>
		</tr>
		<tr>
			<td>ZEN2011 software</td>
			<td>Carl Zeiss Jena GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td colspan="2">Thermo Fisher Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonorex RK 100 SH sonicating water bath</td>
			<td>Bandelin electronic</td>
			<td></td>
			<td>Ultrasonic nominal output: 80 W; Ultrasonic frequency: 35 kHz</td>
		</tr>
	</tbody>
</table>

protocol for microrna transfer into adult bone marrow-derived hematopoietic stem cells to enable cell engineering combined with magnetic targeting

동안 CD133+ 조 혈 줄기 세포 (SCs) 재생 의학 분야에서 높은 잠재력을 제공 하기 위해 입증 되었습니다, 그들의 낮은 유지 비율 관찰된 대규모 세포 사망률 부상된 조직에 주입 후 매우 제한 된 치료 효과입니다. 이러한 한계를 극복 하기 위해 우리가 그들의 관리에 앞서 적당 한 셀 엔지니어링에 대 한 비 바이러스 성 기반된 프로토콜을 확립 하고자 했다. 인간의 CD133 수정+ 셀의 대상 잠재력 뿐만 아니라 통풍 관 효율 및 안전에 관하여 해결 SCs 예측에 관한 (미르) 로드 자석 polyplexes를 사용 하 여 표현. 우리의 프로토콜에 의존, 우리는 CD133 동안 80-90%의 높은 미르 통풍 관 비율을 달성할 수 있는+ 줄기 세포 속성을 받지 않습니다. 또한,이 수정 된 세포는 자기 대상의 옵션을 제공합니다. 우리 여기 CD133의 수정에 대 한 안전 하 고 매우 효율적인 절차 설명+ SCs. 우리는 줄기 세포 치료 효과의 최적화에 대 한 자기 공명 영상 (MRI)을 통해 관리 셀 제품의 모니터링을 위한 표준 기술 제공이 접근을 기대 합니다.

수동 격리와 인간의 BM 파생 CD133의 자석 농축 제시 프로토콜 설명+ SCs 후속 바이러스 독립 셀에 체 외에서 세포 조작 및 에 대 한 비-침략 적 기술 전략, 엔지니어링 vivo 모니터링 도구.
이 3 단계 절연 기술 조밀도 기온 변화도 원심 분리를 통해 미리 소화 sternal BM에서 MNCs의 분리를 허용합니다. 이후에, CD133<...

Watch this Scientific Journal Video about Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting at JoVE.com

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

이 프로토콜에서는 안전 하 고 효율적인 절차 CD133 수정+ 조 혈 줄기 세포. 제시 비 바이러스 성, 자기 polyplex 기반 접근 자기 공명 화상 진 찰을 통해 관리 셀 제품 모니터링에 관해서는 뿐만 아니라 치료 줄기 세포 효과의 최적화를 위한 기반을 제공할 수 있습니다.

성인 골 수 유래 조 혈 줄기 세포로 세포 공학 자기 대상으로 함께 있도록 예측에 관한 전송을 위한 프로토콜

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

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6.5K 조회수.  Rostock University Medical Center. 이 프로토콜에서는 안전 하 고 효율적인 절차 CD133 수정+ 조 혈 줄기 세포. 제시 비 바이러스 성, 자기 polyplex 기반 접근 자기 공명 화상 진 찰을 통해 관리 셀 제품 모니터링에 관해서는 뿐만 아니라 치료 줄기 세포 효과의 최적화를 위한 기반을 제공할 수 있습니다.

비디오: Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

뼈 재생을위한 인적 Perivascular 줄기 세포의 이용

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

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Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

다른 Lineages로 인간 배아 줄기 세포 (hESC)의 전파 및 차별 화를위한 3D 플랫폼으로서 알긴산의 Microcapsule

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

근육 재생의 마우스 모델에서 유도 만능 줄기 세포 유래 메소안지오블라스트 와 같은 근생 성 전구의 이식

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

암 세포의 침략에 응용 프로그램을 사용하여 종양의 기질 미시의 조직 공학

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

셀 라벨 및 초상 자성 산화철 나노 입자로 타겟팅

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

최적화 된 설치 및 히스테리시스 측정 현장에서 자기 도메인 이미징에 대 한 프로토콜

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

내가 조직 공학에 대 한 마우스 Cochleae Decellularizing에 대 한 프로토콜

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

세포가 없는 세균 추출물, 리포좀 및 에멀젼 전달을 사용하여 합성 세포를 생산하는 단백질 을 준비합니다.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

인간 유도 만능 줄기 세포 유래 심근 세포에서 단세포 광학 작용 잠재적 측정

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

정지 인간 조혈 줄기 세포를 유지하는 배양 방법

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...