Name: rapid isolation of bmpr-ib+ adipose-derived stromal cells for use in a calvarial defect healing model
Uploaded: 2017-02-24T13:00:00
Description: Watch this Scientific Journal Video about Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model at JoVE.com

C.D.M. was supported by the American College of Surgeons (ACS) Resident Research Scholarship. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159. M.S.H., H.P.L., and M.T.L. were supported by the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award. H.P.L. was supported by NIH grant R01 GM087609 and a gift from Ingrid Lai and Bill Shu in honor of Anthony Shu. H.P.L. and M.T.L. were supported by the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation. M.T.L. was supported by NIH grants U01 HL099776, R01 DE021683-01, and RC2 DE020771. D.C.W. was supported by NIH grant 1K08DE024269, the Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award.

참고 : 샘플 동의를 주었다 환자를 얻었다. 모든 프로토콜을 검토하고 적절한 스탠포드 대학의 임상 시험 심사위원회의 승인을했다. 인간의 조직과 세포를 처리하는 동안, 항상 당신의 기관에 의해 지정된, 바이오 안전성 레벨 2 (BSL2)주의 사항을 준수합니다. 시약 1. 준비 <ol><li> FACS 버퍼를 준비 : 10 ML의 FBS, 188 5 ㎖ 폴록 사머 500 mL의 멸균 인산 완충 생리 식염수 5 mL의 펜 연?...

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프로토콜 내에서 중요한 단계 의 ASC의 수확하는 동안, 중요한 단계는 콜라겐과 지방의 적절한 소화합니다. 부적절한 소화의 ASC의 낮은 수율 발생합니다. BMPR-IB + 세포를 FACS 정렬 동안 신중 양성하는 게이트를 정의하는 것이 중요하다. 너무 느슨하게 게이트를 정의하는 것은 순수하지 정렬 인구의 원인이 될 수 있습니다. 두개골 결함의 생성 동안, 두개골의 뼈를 통해 결함을 드...

손상, 감염 또는 경부암 인한 주요 골 결손 환자의 회복 및 삶의 질에 큰 영향을 미친다. 다른 기술은 환자의 몸으로부터 뼈 건강 이러한 결함을 채우기 위해 존재하지만,이 이동은 자체 이환율과 합병증 1, 2, 3의 위험도를 전달한다. 또한, 일부 결함이 충분한 기증자 뼈 결함을 채우기 위해 사용할 수없는 너무 크거나 복잡하다. 보철 장치는 뼈 결함을 작성을위한 잠재적 인 옵션이 있지만 이러한 감염 위험, 하드웨어 오류 및 이물 반응 (4) 등 여러 가지 단점과 연결되어 있습니다. 이 때문에, 환자 자신의 세포를 이용한 생물학적 5 골 대체재 공학 가능성에 관심이있다. 지방 유래 기질 세포 (의 ASC)이들은 환자 자신의 지방 조직에서 풍부하게 가능하고 새로운 뼈 조직 (6, 7)를 생성하여 뼈의 결함을 치유 할 수있는 능력을 입증하기 때문에이 애플리케이션에 대한 가능성을 갖는다. 세포의 ASC는 여러 다양한 연구 집단은 특정 세포 표면 마커에 대한 선택하는 향상된 골 형성 활성 (8, 9)와 세포 집단을 생산할 수 있음을 보여 주었다이다. 가장 조골 전위의 ASC를 선택하면 이들 세포 시드 발판 큰 골 결손을 재생할 수있는 가능성을 증가시킬 것이다. 뼈 형태 형성 단백질 (BMP) 시그널링은 골 형성 분화의 ASC (10) 및 (11)에서 골 형성에 중요한 것으로 알려진 BMP 수용체 타입 IB (BMPR-IB)를 조절하는 매우 중요하다. 최근에, 우리는 수 b BMPR-IB의 표현을 보여 주었다전자는 강화 된 골 형성 활동 (12)의 ASC를 위해 선택하는 데 사용됩니다. 여기에서 우리는 생체 내 두개골 결손 모델을 사용하여 골 형성 활동의 분석 뒤에 인간 지방에서의 ASC를 BMPR이-IB가 발현의 분리를위한 프로토콜을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="612">
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			<td height="42" style="height:42px;width:152px;">100 micron cell strainer</td>
			<td style="width:136px;">Falcon</td>
			<td style="width:116px;">352360</td>
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			<td height="21" style="height:21px;width:152px;">24 well plate</td>
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			<td style="width:116px;">3524</td>
			<td style="width:208px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:152px;">40 micron cell strainer</td>
			<td style="width:136px;">Falcon</td>
			<td style="width:116px;">352340</td>
			<td style="width:208px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:152px;">50 mL conical centrifuge tubes</td>
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			<td style="width:116px;">352098</td>
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			<td height="63" style="height:63px;width:152px;">6-0 Ethilon nylon suture, 18&#34;, P-3 needle,&#160;</td>
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			<td style="width:116px;">1698G</td>
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			<td height="42" style="height:42px;width:152px;">Anti-BMPR-IB primary antibody</td>
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			<td>FAB5051A</td>
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			<td style="width:116px;">12565-14</td>
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			<td height="42" style="height:42px;width:152px;">Collagenase Type II powder</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:116px;">17101-015</td>
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			<td style="width:208px;"></td>
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			<td height="42" style="height:42px;width:152px;">Drill: Circular knife 4.0 mm</td>
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			<td style="width:116px;">CK40</td>
			<td style="width:208px;"></td>
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			<td height="42" style="height:42px;width:152px;">Drill: Z500 Brushless Micromotor</td>
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			<td height="21" style="height:21px;width:152px;">FBS</td>
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			<td>10437-077</td>
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			<td height="63" style="height:63px;width:152px;">Fisherbrand Absorbent Underpads, 20&#34; x 24&#34;</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:152px;">Fisherbrand Sterile cotton gauze pad, 4&#34; x 4&#34;</td>
			<td style="width:136px;">Fisher Scientific</td>
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			<td style="width:116px;">DCT-20</td>
			<td style="width:208px;"></td>
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			<td height="42" style="height:42px;width:152px;">Hyclone 199/EBSS medium</td>
			<td style="width:136px;">GE&#160; Life Sciences</td>
			<td style="width:116px;">SH30253.01</td>
			<td style="width:208px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:152px;">Isothesia isoflurane</td>
			<td style="width:136px;">Henry Schein&#160;</td>
			<td style="width:116px;">050033</td>
			<td style="width:208px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Micro Forceps with teeth</td>
			<td style="width:136px;">Roboz</td>
			<td style="width:116px;">RS-5150</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Micro Forceps with teeth</td>
			<td style="width:136px;">Roboz</td>
			<td style="width:116px;">RS-5150</td>
			<td style="width:208px;"></td>
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			<td height="42" style="height:42px;width:152px;">Paraffin film (Parafilm)</td>
			<td style="width:136px;">Bemis</td>
			<td style="width:116px;">PM996</td>
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			<td height="21" style="height:21px;width:152px;">PBS</td>
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			<td style="width:116px;">10010-023</td>
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			<td height="21" style="height:21px;width:152px;">Pen-Strep</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:116px;">15140-122</td>
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			<td height="42" style="height:42px;width:152px;">PLGA scaffolds</td>
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			<td style="width:136px;">Sigma</td>
			<td>P5556-100ML</td>
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			<td height="42" style="height:42px;width:152px;">Polylined Sterile Field, 18&#34; x 24&#34;</td>
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			<td style="width:116px;">696</td>
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			<td>11191</td>
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			<td height="63" style="height:63px;width:152px;">Puralube petrolatum ophthalmic ointment, 1/8 oz. tube</td>
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			<td height="21" style="height:21px;width:152px;">RBC lysis buffer</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:116px;">11814389001</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Webcol alcohol prep swabs</td>
			<td style="width:136px;">Covidien</td>
			<td style="width:116px;">6818</td>
			<td style="width:208px;"></td>
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</table>

rapid isolation of bmpr-ib+ adipose-derived stromal cells for use in a calvarial defect healing model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

마이크로 CT 명확하게 두개골 결함을 보여줍니다 수술 당일에 이루어 스캔. 이 때 4mm 결함에 더 증식가 없을 것입니다. 후속 검사는 기준에 비하여 시간 경과에 따른 결함의 크기를 정량화하는 시간에 걸쳐 얻어진다. BMPR-IB-및 정렬되지 않은 세포 (그림 5)과 비교했을 때 BMPR-IB + 세포와 시드 결함은 결함의보다 신속한 폐쇄를 입증해야한다. 또한, 결함을 포함 ?...

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Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +의 신속한 격리 두개골 결함 치유 모델에서 사용을위한 기질 세포를 지방이는 유래

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

Research

JoVE Journal

Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

분리 및 인간의 농축 향상된 골 연장술에 대한 기질 세포를 지방이 유래

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

연구

발생학

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Automated Quantification of Hematopoietic Cell ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“ Stromal Cell Interactions in Histological Images of Undecalcified Bone

조혈 세포의 자동화 된 정량화 - Undecalcified 뼈의 조직 학적 이미지에서 기질 세포 상호 작용

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

돼지 피하 지방이 조직에서 분리 및 지방이 유래 줄기 세포의 분화

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

지방 창고 별 SCA1의 면역 자기 분리<sup&gt; 높은</sup&gt; 지방이 유래 줄기 세포 (의 ASC)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

인간 태반 조직에서 추출 분리 및 중간 엽 줄기의 확장 / 기질 세포

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

원심 중력에 의해 지방이 유래 줄기 세포의 연골 분화 유도

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

마우스 자궁 내막 상피와 기질 세포의 분리에 대한<em&gt; 체외</em&gt; 탈락

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

인간 제대혈 및 태아 태반에서 중간 엽 기질 세포의 분리 및 특성

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

Vivo에서 인간 지방 유래 중간 엽 줄기 세포 형광 질 성 막 염료와 쥐 무릎 관절염 모델에서의 추적

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

생체 외에서 Decidualization에 대 한 인간의 자궁내 막 기질 세포의 고립

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...