이 작품은 국립 보건원 (DBC에 HL068915), (CAP로) 채찍질 연구 기금에서 새 연구원 수상에서 연구 보조금, 보조금의 에이드 (DBC에 12GRNT11910008) 미국 심장 협회 (American Heart Association)에서보기에 의해 지원되었다 , 어린이 심장 재단 (DBC에), 그리고 보스턴 아동 병원 심장 전도 기금, 라이언 가족 성기 사이즈, 그리고 데이빗 풀먼에 의해 기부에서 연구 보조금.

여기에서 우리는 어린 양의 피부에서 COL1의 분리를 보여줍니다. 서면으로,이 프로토콜은 또한 성공적으로 토끼와 인간의 피부로부터 COL1를 분리하는데 사용될 수있다. 1. 피부 샘플을 준비 <ol><li> 사용하기 전에 4 ° C에 모든 시약을 평형. </li><li> 얼음처럼 차가운 dH보다 2 O의 피부 샘플 (25~50g)을 씻어 제모 크림 어떤 울, 모피, 또는 머리를 제거합니다. </li><li> 결합 조직과 지방의 샘플 깨끗하게 긁어 단일 에지 면도날을 사용합니다. </li><li> 얼음처럼 차가운 dH보다 2 O.에서 샘플을 씻어 </li><li> 단일 에지 면도날 1cm X 1cm 조각으로 피부 샘플을 조각. </li></ol> 2. Noncollagenous 가용화 물질을 제거 <ol><li> 샘플 50 ㎖ 당 원뿔 튜브의 5g을 달아 얼음처럼 차가운 0.5 M 아세트산 나트륨 30 ㎖를 추가합니다. </li><li> 벤치 탑 균질의 50 ML 튜브 어댑터를 사용하여 6m / 초 설정으로 1 분 동안 튜브를 섞는다. </li><li> SUP 폐기7 아세트산 나트륨 세척주기의 총 ernatant를 반복합니다. </li><li> 얼음처럼 차가운 dH보다 2 O에서 샘플을 씻어 잔류 아세트산 나트륨을 제거하기 위해 한 번 섞는다. </li><li> 물기를 제거하고 신선한 50 ML 원뿔 튜브로 전송 튜브의 측면에 대한 샘플을 압축하기 위해 주걱을 사용합니다. </li></ol> 3. I 콜라겐 추출을 입력 <ol><li> 샘플을 압축하고 각 세척 후 상층 액을 버리고 벤치 탑 균질 6 M / 초에서 1 분에 대한 ㎖ / g의 2 배의 샘플을 씻어 0.075 M 시트르산 나트륨 완충액. </li><li> 샘플에 0.075 M 시트르산 나트륨 완충액의 새로운 2 ㎖ / g 나누어지는을 추가합니다. </li><li> 각주기 사이의 버퍼를 제거하지 않고 6 연속 1 분, 교반 6m / 초 벤치 탑 균질 혼합주기를 수행합니다. </li><li> 포집 관에 두꺼운, 상등액을 전송합니다. </li><li> 샘플 ㎖ / g (1 건) 0.075 M 시트르산 나트륨 완충액을 추가하고 마지막으로 벤치 탑 균질화 AGITA을 수행기의 순환. </li><li> 포집 관이 최종 뜨는을 추가합니다. </li><li> 4 ℃에서 10 분 동안 3,200 XG에서 포집 관을 원심 분리기 </li><li> 100,000 분자량의 톱 수 납부에 뜨는을 전송 원심 필터 장치를 잘라. </li><li> 4 ℃에서 30 분 동안 3,200 XG에 원심 분리기 </li><li> 4 ° C에서 깨끗한 원뿔 관 및 저장에 상부 구획으로부터 정제 된 COL1 이동 </li></ol>

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저자는 공개 할 경쟁 금전적 이해 관계가 없습니다.

이 방법의 중요한 구성 요소는 물기를 제거하는 피부 샘플의 반복 효율적인 압축입니다. 이 샘플을 압축하여 단계 2.5에 극력 아세트산 나트륨을 제거하는 것이 중요하다. 우리는 튜브의 측면에 대해 피부를 압축 주걱을 사용하지만, 무명천도 사용될 수, 이것은 튜브에서 샘플의 제거를 필요로하고이 단계를 수행하는 데 필요한 시간을 증가시킬 것이지만. 마찬가지로, 시트르산 나트륨의 양이 후?...

수십 년 동안, 연구자 및 상업 벤더 사용할 수 있습니다 조직 COL1 원 섬유의 매트릭스의 재 부유 결과 중화 다음에 간단한 산 추출 프로토콜의 일부 변화를 이용하여 피부와 힘줄 등의 조직 소스의 구색에서 용해 COL1을 격리했다 생물 의학 응용 프로그램 1-4의 다수를위한. COL1에 대한 임상 응용의 많은 사례가 있지만이 단백질의 준비 5-7을 수행하는 일 또는 주를 복용 긴 추출을 필요로하기 때문에, 이들 중 몇몇은 autologously 파생 COL1를 사용합니다. 그 결과, 연구 조사와 의사는 일반적으로 쥐, 소 또는 돼지의 진피와 같은 인간이 아닌 조직을 사용하거나 시체에서 제거 스킨을 사용하거나 남성 할례에 따라 제조하는 COL1의 비용, 상업 준비를 사용합니다. 재생 응용 프로그램의 경우, 자신의 능력에 대한 이러한 제품의 전시 변화의 대부분은 고체 형성매트릭스 또는 세포 부착 및 성장을 지원할 수있는 조직 구조. 따라서, 신속하게 접근 할 수 있고 충분한자가 조직 소스에서 COL1를 추출하기 위해 디자인 된 새로운 표준화 된 방법에 대한 구별이 필요하다. 이러한 방법은 COL1은 또한 동물 모델에서 추출 콜라겐 계 지지체에 조립 설계 조직의 전임상 시험에 사용될 수 연구 설정에 시간과 비용을 절약한다. 동시에, 병원에서 급속 절연 autologously 유래 COL1를 사용하는 옵션을 갖는, 그렇지 않으면 동종 또는 이종 제제를받을 환자의 전체적인 안정성을 증가시킬 것이다. autogeneic 제제를 투여받은 환자의 경우,이 간소화 된 방법은 크게 생검 수집 및 콜라겐 애플리케이션 간의 간격을 줄일 것이다. 이러한 이유로, 우리는 고속 AG를 사용하여 노포 COL1 분리 및 정제 방법을 개선하고자itation 및 크기 배제 원심 분리. 이 방법은 많은 실험실에서 발견 된 표준 버퍼 및 장비를 사용하여 수행하는 것이 간단합니다. 고속 교반을 사용함으로써, 우리는 프로토콜 미만 3 시간 (8)에 대략 10 일 ~ 용해, 경피 COL1을 분리하는 데 필요한 시간을 감소시킨다. 중요한 것은,이 방법은 쉽게 절차의 단일 세트 동안 환자에 사용 autologously 유래 COL1을 준비하기 위해 임상 환경에서 수행 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="544">
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		<col />
		<col />
		<col />
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	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
			<td style="width:147px;">116004500</td>
			<td style="width:139px;">-</td>
		</tr>
		<tr height="190">
			<td height="190" style="height:190px;width:123px;">Centrifuge</td>
			<td style="width:136px;">Beckman</td>
			<td style="width:147px;">J6-MI</td>
			<td style="width:139px;">Swing bucket rotor to accommodate 50 mL conical tubes at 3200 g.</td>
		</tr>
	</tbody>
</table>

an improved method for the preparation of type i collagen from skin

용해 1 형 콜라겐 (COL1)는 세포 배양을위한 접착제 기판으로 회생 응용 프로그램을위한 세포 지지체로 광범위하게 사용됩니다. 임상 적으로,이 단백질 널리 성형 수술, 피부 주사, 골 이식 및 재건 수술에 사용됩니다. 이러한 절차에 대한 COL1의 소스는 염증과 거부에 대한 가능성뿐만 아니라 생체 이물 질병의 전송을 증가하는, 일반적으로 인간이 아닌 있습니다. 이러한 관점에서, 효율적이고 신속 autologously 유래 조직의 한정된 양에서 COL1을 정화하는 방법은 많은 문제점을 회피 할하지만, 표준 격리 프로토콜 긴이며 종종 콜라겐 조직의 대량을 요구한다. 여기서, 우리는 이하의 3 시간에 약 10 일에서이 단백질을 분리 및 정제하는 데 필요한 시간을 줄여 효율적인 COL1 추출 방법을 설명한다. 우리는 많은 수술 과정 중에 있기 때문에 가능 여부에 대한 우리의 조직​​ 소스로 진피를 선택했다. 티그의 방법은 진피의 적은 양의 고순도, 수용성 COL1을 얻기 위해 강력한 교반 및 원심 여과와 함께 기존의 추출 버퍼를 사용합니다. 간단히, 피부 조직 검사는 지방, 결합 조직, 그리고 머리를 제거한 후 얼음처럼 차가운 dH보다 2 O 철저히 세척. 피부 샘플은 0.5 M 아세트산 나트륨 및 고속 벤치 탑 균질화기를 사용하여 단백질 및 다당류 noncollagenous 스트리핑된다. 잔류 고형물로부터 콜라겐이어서 균질화기를 사용하여 0.075 M 시트르산 나트륨 완충액으로 추출 하였다. 이 추출물을 비교하거나 우수한 상용 제품에 대한 품질이나 기존의 프로 시저를 사용하여 얻은 결과의 COL1 준비를 얻을 수 100,000 MW 컷 - 오프 원심 필터를 사용하여 정제된다. 우리는이 방법은 연구와 임상 응용 프로그램의 다수에 대한 autologously 파생 COL1의 활용을 용이하게 기대하고 있습니다.

이 COL1 격리 프로토콜은 완료하는 데 약 2 시간 15 분을 필요로한다. 그림 1은이 절차의 주요 단계를 요약 한 개략도를 보여줍니다. 샘플 준비 및 고속 동요의 12 사이클을 수행하고 아세트산 나트륨 헹굼은 약 35 분 (도 1a-C)가 소요된다. 한 교반을 수행하고 dH보다 2 O와주기를 씻어은 약 5 분 (그림 1D 및 1E)가 소요된다. 구연산 나트륨을 추가 한 교반에 샘...

Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

An Improved Method for the Preparation of Type I Collagen From Skin

수용성 제 1 형 콜라겐 (COL1)의 분리에 대한 기존의 절차 때문에 긴 버퍼 배양 및 섬유의 힘든 resuspensions의 처음부터 끝까지 약 10 일이 필요합니다. 여기, 우리는보다 3 시간에 작은 피부 생검에서 COL1을 정화 할 수있는 방법을 설명합니다.

피부에서 타입 I 콜라겐의 제조를위한 개선 된 방법

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

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22.7K 조회수.  Boston Children's Hospital.  수용성 제 1 형 콜라겐 (COL1)의 분리에 대한 기존의 절차 때문에 긴 버퍼 배양 및 섬유의 힘든 resuspensions의 처음부터 끝까지 약 10 일이 필요합니다. 여기, 우리는보다 3 시간에 작은 피부 생검에서 COL1을 정화 할 수있는 방법을 설명합니다. 

비디오: An Improved Method for the Preparation of Type I Collagen From Skin

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotypic 콜라겐 I 분석 : 3 차원 컨텍스트에서 셀 행동을 평가하기 위해 가단 플랫폼

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

연구

생체공학

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

방법 혈액 - 뇌 장벽의 인간 세포 기반의 연락 모델의 준비를 위해 개선

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

뼈 이식 앵커리지 평가하는 향상된 기계적 시험 방법

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

압축 된 콜라겐에 다진 조직 : 단일 무대 재건 복구를위한 세포 함유 Biotransplant

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Keratinocyte 확산에 2-및 3 차원의 평가 유형 I 교원 질 기판

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

암 세포 배양에서 수집된 매체에서 액체 크로마토그래피 질량 분광법에 의해 분석된 선택된 Kynurenines의 동시 정량화

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

조직 손상을 줄이기 위한 새로운 최소 침습 느린 주입 방법

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

저자 스포트라이트: 최소 침습을 위한 새로운 세포 주입 방법  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

침습을 최소화하는 새로운 세포 주입 방법

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...