저자 실용적인 조언을 제공 하는 학과의 플라스틱 및 재건 수술, 준 텐도 대학의과 대학, 박사 유키코가 감사 합니다. 우리는 또한 당뇨병 센터의 도쿄 여자의 의학 대학의과 대학 우수 기술 지원의 씨 카 즈 무라를 감사합니다. 이 연구는 교육, 문화, 스포츠, 과학 및 기술 (MEXT) 일본에서 개발 혁신 시스템 "셀 시트 조직 공학 센터 (CSTEC)" 프로젝트의 학 제 연구 분야 프로그램 고급 혁신 센터의 창조에 의해 지원 되었다.

All experimental protocols presented below were approved by the Animal Welfare Committee of Tokyo Women&#39;s Medical University School of Medicine and abided by all requirements of the Guidelines for Proper Conduct of Animal Experiments.
1. Preparation of Animals, Instruments, Culture Media, and Dishes
<ol>
	<li>Prepare complete culture medium using minimum essential medium alpha containing 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Store this for several months at 4 &#176;C until use.</li>
	<li>Use adult male ZDF rats as a type 2 diabetic obesity model. Use the fat pad of male Lewis rats to isolate adipose tissue to prepare the cell sheets.</li>
</ol>
2. Isolation and Culture of rASCs
<ol>
	<li>Obtain adipose tissue from Lewis rats (8 - 33 weeks old, male) under general anesthesia using isoflurane.
	<ol>
		<li>Prepare a rodent mechanical ventilator and 4% isoflurane. Prepare several sterile cotton tips, clean gauzes, a scalpel, a needle holder, operating scissors, a pair of forceps, and a 5-0 nylon suture. Sterilize the surgical instruments and supplies.</li>
		<li>Prepare a 100-mm Petri dish with 10 mL of sterile phosphate-buffered saline (PBS) to temporarily preserve the obtained adipose tissue. Weigh the Petri dish with PBS using a balance before starting surgery to measure the collected adipose tissue. Lay out the surgical instruments and supplies on a sterile drape.</li>
		<li>Anesthetize the rats by using isoflurane at 3 - 4% via a rodent mechanical ventilator and maintain the rats at 2 - 3% isoflurane during surgery. Monitor the depth of anesthesia by observing the depth and rate of respiration of each rat.</li>
		<li>While wearing sterile gloves, position the rat in a supine position on a sterile drape. Shave the operative area with an electric razor and clean the abdominal section of the rat using sterile gauze soaked in 70% ethanol.</li>
		<li>Create an approximately 5 cm-long skin incision with a scalpel in the lateral lower abdomen of the rat. Expose and dissect the external oblique muscle. Then, expose the epididymal adipose tissue surrounding the epididymis. Gently pull the epididymal adipose tissue out. 
		NOTE: The adipose tissue can be obtained on either side of the rat&#39;s abdomen.</li>
		<li>Excise only the epididymal adipose tissue, avoiding damage to the epididymis, testis, and epididymal blood vessels (Figure S1). Soak the excised adipose tissue in the PBS in the Petri dish to prevent dryness and weigh the tissue.</li>
		<li>Close the abdominal muscle with 5-0 VICRYL and the skin with a 5-0 nylon suture. Then, return each rat that has undergone surgery to a separate cage (one rat per cage). 
		NOTE: Monitor the rats until they fully recover.</li>
	</ol>
	</li>
	<li>Isolate rASCs from 2 - 3 g of adipose tissue (excised from a Lewis rat and processed according to a previously-reported method)27. Briefly, enzymatically digest the excised adipose tissue with 0.1% type A collagenase at 37 &#176;C for 1 h to obtain the stromal-vascular fraction (SVF; Figure S2).
	<ol>
		<li>Prepare several 100-&#181;m cell strainers, several 50-mL tubes, several 15 mL tubes, and two scalpels on a biological clean bench. Turn on a centrifuge and warm up a water bath to 37 &#176;C. In addition, prepare approximately 50 mL of sterile PBS in a 50 mL tube with 0.1% (final concentration) of type A collagenase.</li>
		<li>Mince the adipose tissue into small pieces using two scalpels.</li>
		<li>Move all minced adipose tissue to a 50-mL tube and add PBS to bring the total volume to 15 mL.</li>
		<li>Rest the 50-mL tube upright for 5 min. Discard the upper layer and collect the lower layer except for the debris.</li>
		<li>Add 0.1% of type A collagenase solution to the 50-mL tube with PBS containing the upper layer in order to enzymatically digest the adipose tissue. 
		NOTE: Warm approximately 20 mL of PBS per 2 - 3 g of adipose tissue in a 37 &#176;C water bath.</li>
		<li>Tilt and shake the 50 mL tube gently at 120 - 130 rpm for 1 h in a 37 &#176;C water bath.</li>
		<li>Rest the 50 mL tube upright for 5 min after shaking.</li>
		<li>Filter the solution using a 100 &#181;m cell strainer and pour the solution into a new 50 mL tube. Dispense the filtered solution into two 15 mL tubes.</li>
		<li>Centrifuge the solution in the 15 mL tubes for 5 min at 700 x g. Carefully remove the upper layer of supernatant and leave 5 mL of the lower layer of the solution. Do not aspirate the pellet.</li>
		<li>Add approximately 5 mL of complete culture medium to each 15 mL tube, up to 10 mL per 15 mL tube, and carefully re-suspend the pellet.</li>
		<li>Repeat steps 2.2.8 - 2.2.9. Then, obtain the SVF.</li>
	</ol>
	</li>
	<li>Prepare two 60 cm2 culture dishes. Collect the SVF after two centrifugations at 700 &#215; g for 5 min. Suspend the SVF and plate 10 mL of SVF solution on each 60-cm2 culture dish.</li>
	<li>Culture the dishes for 24 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Prepare PBS and 0.25% trypsin-ethylenediamine tetraacetic acid (EDTA), storable for several months at 4 &#176;C until use.</li>
	<li>After initial plating (24 h), wash the cells with PBS 3 times to remove unattached cells and debris. Add fresh complete medium.</li>
	<li>Passage the cells that are nearly sub-confluent on day 3. Add 1 mL of 0.25% trypsin-EDTA and incubate the cells at 37 &#176;C in a 5% CO2 incubator for 3 - 5 min. 
	NOTE: Complete the trypsinization step within 10 min to prevent cell damage.</li>
	<li>Observe the cells under a light microscope after trypsinization to confirm that all the cells have thoroughly detached from the dishes. Then, add 9 mL of complete culture medium to the dishes.</li>
	<li>Count the number of cells using a hemocytometer.</li>
	<li>Subculture the cells at a density of 1.7 x 103 cells/cm2 every 3 days until passage 3. Culture the subcultured cells at 37 &#176;C in the humidified atmosphere of a 5% CO2 incubator.</li>
</ol>
3. Creation of rASC Sheets
<ol>
	<li>Prepare several 35-mm diameter temperature-responsive culture dishes. Pre-coat the dishes with FBS (or complete medium containing FBS) at 37 &#176;C in a 5% CO2 incubator for more than 1 h. 
	NOTE: A 35 mm diameter temperature-responsive culture dish is a commercially available product.</li>
	<li>Prepare a thermo-plate and incubate at 37 &#176;C for the following experimental procedures. 
	NOTE: Perform every procedure using temperature-responsive culture dishes on a 37 &#176;C thermo-plate to prevent the cells from spontaneously detaching from the dish.
	<ol>
		<li>Warm the complete culture medium used in the procedures to 37 &#176;C prior to experimentation.</li>
		<li>Seed passage 3 rASCs derived from Lewis rats onto a 35 mm diameter temperature-responsive culture dish at a density of 1.5 x 105 cells/dish for 3 days using a 2 mL total volume of complete culture medium. 
		NOTE: When rASCs are plated onto a new culture dish, the dish should be slowly rocked back and forth and left and right in an incubator to achieve uniform rASC seeding and a uniformly thick rASC sheet.</li>
		<li>Change 2 mL of the medium to 2 mL of complete culture medium containing 16.4 &#181;g/mL L-ascorbic acid phosphate magnesium salt n-hydrate (AA) on day 3 after seeding to the temperature-responsive culture dishes incubated on a 37 &#176;C thermo-plate. 
		NOTE: Dissolved AA can be stored at -30 &#176;C for months.</li>
		<li>Culture the cells for an additional 4 - 5 days and replace the medium every 2 days with fresh medium containing AA.</li>
		<li>Confirm the proliferation and generation of ASCs under a light microscope to determine whether gaps occurred between the cells in a contiguous cell sheet.</li>
	</ol>
	</li>
	<li>Transfer the cell sheets from the incubator to the benchtop for 15 - 20 min to cool them to room temperature.; observe the cells spontaneously detach as a contiguous cell sheet. Harvest the rASC sheet from the dish surface with a pair of forceps. 
	NOTE: Usually, rASC sheets can be handled with a pair of forceps. If necessary, a transfer membrane can be used to transfer a cell sheet from the culture dish to the wound site if the cell sheet is brittle and fragile.</li>
</ol>
4. Preparation of the Full-thickness Skin Defect Wound Model and Transplantation of rASC Sheets
<ol>
	<li>Prepare a rodent mechanical ventilator and 4% isoflurane. Prepare several sterile cotton tips, clean gauze, 5-0 nylon suture, a scalpel, a periosteal raspatory, a needle holder, operating scissors, and a pair of forceps. Sterilize the surgical instruments and supplies. Lay out the surgical instruments and supplies on a sterile drape.</li>
	<li>Prepare PBS and maintain it at room temperature, along with some artificial skin and a non-adhesive dressing. Precut the artificial skin (15 x 10 mm) and non-adhesive dressing (20 &#215; 15 mm). Soak the inner collagen sponge layer of the artificial skin in saline before using the artificial skin. 
	NOTE: The artificial skin is made of two layers: an outer silicone sheet layer and an inner collagen sponge.</li>
	<li>Use ZDF rats (16 - 18 weeks old, male, 500 - 600 g) as a wound-healing model for type 2 diabetes and obesity.</li>
	<li>Anesthetize the ZDF rats by inhalation of 4% isoflurane using a rodent mechanical ventilator. Induce anesthesia by using isoflurane at 4 - 5% via a rodent mechanical ventilator and maintain it at 3 - 4% during surgery. Monitor the depth of anesthesia by observing the depth and rate of respiration of each rat via a toe pinch.</li>
	<li>While wearing sterile gloves, position the rat in the prone position on a sterile drape. Clean the head of the rat using sterile gauze soaked in 70% ethanol, shave the operative area with an electric razor, and clean the skin after shaving using sterile gauze soaked in 70% ethanol.</li>
	<li>Create a squared full-thickness skin defect (15 x 10 mm2) on the head of the anesthetized ZDF rats by removing the cutaneous tissue from the epidermis to the periosteum. Excise the skin and cutaneous tissue with a scalpel and remove the periosteum with a periosteal raspatory.</li>
	<li>Move the rASCs on the 35 mm temperature-responsive culture dish from the incubator to a room-temperature environment immediately prior to transplantation.</li>
	<li>Observe the rASCs on the 35 mm temperature-responsive culture dish spontaneously detach as a sheet from the dish surface after the formation of a wound. Harvest the rASC sheets after 7 days of culturing on temperature-responsive culture dishes. Remove the cultured medium and wash the rASC sheet with PBS three times. Soak the rASC sheet in PBS until transplantation to prevent desiccation.</li>
	<li>Place the rASC sheet immediately over the skull defect using a pair of forceps. If the cell sheet is brittle and fragile, a membrane can be used as a scaffold for transferring the cell sheet from the culture dish to the wound site.</li>
	<li>Cover the rASC sheet and defect with the artificial skin (15 x 10 mm2) and close the wound with approximately 10 stiches using the 5-0 nylon suture. To protect the wound, place a non-adhesive dressing (20 x 15 mm2) over the artificial skin, using 5-0 nylon sutures to keep the wound environment moist and to absorb exudates. 
	NOTE: The non-adhesive dressings will be removed by the ZDF rats within a few days after application. Therefore, it is necessary to regularly monitor the rats after transplantation. Usually, the non-adhesive dressing is replaced every 2 days under general anesthesia.</li>
	<li>Return each rat that has undergone surgery to a separate cage until they have fully recovered (one rat per cage). After an observation period, euthanize the rats using the approved method of isoflurane overdose.</li>
</ol>

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다음 저자 공개할 금융 관계가이 게시에 관련: 男 카노는 설립자와 셀 씨 주식 회사, 기술 및 도쿄 여자의과 대학에서 특허 라이센스, 위원회의 감독 이며 男 오 카노 마사유키 야마토 있으며 셀 씨 주식 회사 도쿄 여자 의과대학 관계자 받는 셀 씨 i n c.에서 연구 자금 다른 저자는 그들은 금융 관계가이 게시에 관련 없는 선언 합니다.

성공적으로 경작 된 rASC 시트에 대 한 가장 중요 한 단계는 다음과 같습니다: 1) 온도 약 37 ° C에서 온도 응답 문화 요리에 경작 하는 동안 유지 되어야 합니다. RASC 시트를 생성 하는 동안 모든 절차 37 ° C 온도-접시에, 수행 하 고 모든 시 약은 저절로 요리31에서 분리에서 세포를 방지 하기 위해 37 ° C로 예 열. 2) 받는 사람에서 쥐 rASC 시트의 성공적인 이식에 대 한 중요 한 비 접착...

당뇨병 환자의 인구 세계 전반 증가 하 고 20151;에서 400 백만 도달 당뇨병 환자의 약된 15-25% 더 낮은 말단 당뇨병 궤 양2의 진행에서 위험에 있습니다. 더 낮은 말단 당뇨병 궤 세공 되지 않으며 완전 한 회복 후 재활 훈련으로 장기간 치료를 해야 할 수도 있습니다. 긴 치료 기간을 종종 환자 삶의 질에 있는 뜻깊은 감소에 있는 결과. 따라서, 당뇨병 상처 치료에 대 한 감소 또는 악화를 방지 하는 새로운 치료를 개발 해야 합니다. 당뇨병 상처 치유를 평가 하려면 우리는 당뇨 궤 양 상처 치유 모델 쥐, 모방 실용적인 임상 조건, 최적화 그리고 치유 상처 가속 지방 유래 줄기 세포 (ASC) 시트 셀 시트 엔지니어링을 사용 하 여 이식 여부 평가.
Mesenchymal stromal 세포 (MSCs) 그들의 자기 갱신, 그들의 immunomodulatory 효과 용량과 다양 한 세포 계보3으로 차별 하는 그들의 능력 때문에 상처 치유를 가속을 위한 우수한 잠재력을 전시. ASCs는 지방 조직에서 파생 된 MSC의 유형 그리고 그들은 그들의 잠재적인 신생 및 paracrine 활동4,5를 포함 하 여 다른 조직에서 파생 된 MSCs에 몇몇 이점을 전시. 지방 조직, 인체에서 상대적으로 풍부한 이며 최소한 침략 적 절차를 사용 하 여 컬렉션에 대 한 접근 허용. 따라서, ASCs 상처 치유 응용 프로그램6,7에 대 한 실험적으로 사용 되었습니다.
이전 보고서 상처 주변 지역에 단일 셀 MSC 정지 직접 주입 상처 치유8,9를 가속화할 수 있습니다 나타났습니다. 그러나,이 당뇨병 궤 양 모델 단일 세포 현 탁 액의 주입에 따라 상처 치유의 가속의 보고서에도 불구 하 고 상처 사이트에서 이식된 세포의 생존에 명확 하지 않습니다.
이 연구에서 우리가 온도 응답 문화 요리를 사용 하 여 셀 시트 공학 적용. 이 요리는 온도 응답성 고분자 N-isopropylacrylamide covalently 그들의 표면10에 바인딩된. 이식할된 폴리머 레이어를 온도-제어 세포 접착 또는 문화 접시의 표면에서 분리 수 있습니다. 접시의 표면이 된다 37 ° C에서, 반면 세포 표면에서 32 ° c 온도에서 친수성이 되 면 저절로 분리 준수 및 증식, 세포 수 소수 배양된 세포 온도; 감소 하면 그대로 셀 접합 및 세포 외 매트릭스 (ECMs)와 인접 셀 시트 수확 수 있습니다. 따라서, ECM, 트립 신, 등을 손상 하는 분해 효소 필요11않습니다. 따라서, 셀-시트 공학 셀 연결을 유지 하 고 세포 이식의 효능을 향상 시킬 수 있습니다.
또한, 셀-시트 이식 세포 주입12에 비교 될 때 세포 생존 율을 증가 시킵니다. 이 프로토콜에서 커 당뇨 지방 (ZDF) 쥐 지연된 상처 치유와 타입 2 당뇨병과 비만 모델 선정 됐다. 쥐 자발적으로 약 4 주에 비만 개발합니다. 그들은 다음 어느 시점에서 그들은 혈당 인슐린 저항, dyslipidemia, 그리고 hypertriglyceridemia13과 관련 된 전시 하는 나이의 8과 12 주 사이 비만과 2 형 당뇨병을 개발. 상처 지연된 치유, 주변 혈관, 당뇨병 신 장병에 감소 된 혈은 또한14,,1516을 관찰 했다. 또한, ZDF 쥐 다루기 힘든 피부 궤 양, 당뇨 궤 양 등의 치유 공부에 대 한 적절 한 모델을 수 있습니다.
상처 치유 메커니즘에 인간과 설치류의 차이 피부에 해 부 차이와 연결 됩니다. 정상적인 쥐에서 치유 하는 상처는 다시 epithelialization 및 알갱이 만듦 조직 형성에 따라 인간의 상처 치유 하는 반면 상처 수축, 기반. 일반적으로, 상처 부 설치류 모델에 사용 되 상처 수축 최소화 하는 데 도움이 하 고 비록 nondiabetic 쥐에 상처 수축에 의해 거의 완전히 폐쇄는 알갱이 만듦 조직17, 점차적인 형성에 대 한 수 있습니다. 그러나, 당뇨병 상처 수축 ZDF에서 쥐 장애인, 그리고 상처 치유 주로 발생 통해 다시 epithelialization 및 알갱이 만듦 조직 형성; 따라서,이 과정은 인간의 상처 치유14더 비슷합니다.
Debridement 임상 발생 종종 후 당뇨병 상처를 노출 된 뼈. 12 m m 직경 athymic 누드 마우스18,19 의 뒤에 전체 간격 피부 상처와 정상적인 쥐20의 뒤에 10 m m 직경 전체 간격 피부 상처 이전 연구 조사가 있습니다. 심한 당뇨병 상처에 대 한 임상 모델을 개발, 큰 (15 x 10 m m2) 전체 간격 피부 결함 뼈를 노출 하 고는 하지 않고 만들어졌습니다, 앞에서 설명한21, 제 2 형 당뇨병 및 비만 쥐에서.
일반 루이스 쥐의 ASCs에서 쥐 ASC (rASC) 시트는 ASC 시트 allogenic 이식 통해 창조 되었다. 임상 연습에서 궤 양은 당뇨병 환자 자주 전시 통제 높은 혈액 포도 당 수준 및 높은 몸 질량 색인, 이러한 환자에서 지방이 많은 직물을 얻기의 어려움을 증가 하는 이러한 합병증 원인 상처 치유 장애 등 심각한 당뇨 합병증 때문에 헌 식은 가능 하지입니다. 또한, 당뇨병 전시회와 동물에서 ASCs 속성을 변경 하 고 기능22장애인. 따라서, 여기에 제시 된 프로토콜 정상 쥐에서 rASC 시트 allogenic 이식 및 당뇨병 쥐에 인공 피부의 응용 프로그램 설명 합니다.
이 프로토콜에서 사용 하는 bilayer 인공 피부 상처의 자발적 수축 방지, 새로운 결합 조직 매트릭스의 합성을 촉진과 진정한 피23를 닮았다. 이 프로토콜에서 인공 피부는 rASC 시트에 배치 하 고 상처 수축 또는 느슨한 쥐 피부에서 발생 하는 확대를 방지 하기 위해 나일론 스레드 고정. 또한, 인공 피부 ASC 시트에 대 한 3 차원 프레임 워크를 제공, 이식 ASC 시트 및 상처, 습 한 환경 유지와 감염과 외부 세력 으로부터 상처를 보호 합니다. 마지막으로, 비 접착 드레싱 외부 충격에서 그것을 보호, 촉촉한 상처 환경을 유지 및 흡수 exudate 상처 위에 배치 됩니다.
RASC 시트, 유연한, 얇고 변형 이며 박동 심장24같은 받는 사람 사이트 준수 수 있습니다. 셀-시트 엔지니어링과 다양 한 조직 재건을 위해 사용 되어 치료 효과25,26. 를 생성할 수 있습니다. 임상 치료 잠재력을 전시 하는 ASC 시트의 다양 한 상처 치유 가속 수 있습니다. 또한, ASC 시트, 인공 피부의 사용과 결합의 수용자 이식 다루기 힘든 궤 양이나 화상, 말 초 동맥 질환 또는 교원 질 질병, 관찰 등의 치료에 적용할 수 있습니다 또는 영양 부족은 또는 스테로이드를 사용 하는 환자에 게 관리 될 수 있습니다. 이 방법은 ASCs 이식의 효율성을 증가 시킵니다. 상처 치유에서 쥐 모델 인간의 상처 치유 과정을 닮 고 소형 실험 동물 임상 상태를 모방 하는 심각한 상처 상태를 생성 합니다.

<table><tbody><tr><td>&amp;alpha;-MEM glutamax</td><td>Invitrogen</td><td>32571-036</td><td>Carlsbad, CA</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Japan Bioserum Co Ltd.</td><td>S1650-500</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Collagenase A</td><td>Roche Diagnostics</td><td>10 103 578 001</td><td>Mannheim, Germany</td></tr><tr><td>60 cm&lt;sup&gt;2&lt;/sup&gt; Primaria tissue culture dish</td><td>BD Biosciences</td><td>353803</td><td>Franklin Lakes, NJ</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffer Saline (PBS)</td><td>Life Technologies</td><td>1490-144</td><td></td></tr><tr><td>0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>L-ascorbic acid phosphate magnesium salt n-hydrate</td><td>Wako</td><td>013-19641</td><td></td></tr><tr><td>35-mm temperature-responsive culture dish (Upcell&lt;sup&gt;TM&lt;/sup&gt;)</td><td>CellSeed</td><td>NUNC-174904</td><td>Tokyo, Japan</td></tr><tr><td>Microwarm plate (MP-1000)</td><td>Kitazato Science Co., Ltd.</td><td>1111</td><td></td></tr><tr><td>Rodent mechanical ventilator</td><td>Stoelting</td><td>#50206</td><td>Wood Dale, IL</td></tr><tr><td>4% isoflurane</td><td>Pfizer Japan</td><td>114-13340-3</td><td>Tokyo, Japan</td></tr><tr><td>Artificial skin (Pelnac&lt;sup&gt;&amp;reg;&lt;/sup&gt;)</td><td>Smith &amp;amp; Nephew</td><td>PN-R40060</td><td>&amp;nbsp;Tokyo, Japan</td></tr><tr><td>Non-adhesive dressing (Hydrosite plus&lt;sup&gt;&amp;reg;&lt;/sup&gt;)</td><td>Smith &amp;amp; Nephew</td><td>66800679</td><td>Known as Allevyn non-adhessing&lt;sup&gt;&amp;reg;&lt;/sup&gt; in the United State</td></tr><tr><td>5-0 nylon suture</td><td>Alfresa</td><td>EP1105NB45-KF2</td><td></td></tr><tr><td>20 CELLSTAR TUBES</td><td>greiner bio-one</td><td>227 261</td><td></td></tr><tr><td>15 mL Centrifuge Tube</td><td>Corning Incorporated</td><td>430791</td><td></td></tr><tr><td>14 GOLDMAN-FOX PERIOSTEAL</td><td>Hu-Friedy</td><td>P14</td><td>Chicago, IL</td></tr></tbody></table>

creation and transplantation of an adipose-derived stem cell (asc) sheet in a diabetic wound-healing model

인공 피부는 임상 연습에 상당한 치료 결과 달성 했다. 그러나, 당뇨병 환자 비능률적된 혈 또는 큰 상처와 상처에 대 한 인공 피부 치료 연장 될 수 있습니다. 세포 기반 치료 당뇨병 궤 양의 치료에 대 한 새로운 기술로 등장 하 고 셀 시트 공학 세포 이식의 효능을 향상 했다. 보고서의 수는 지방 유래 줄기 세포 (ASCs), 중간 엽 기질 세포 (MSC), 유형의 지방 조직 및 다른 조직에서 MSCs에 비교 될 때 컬렉션에 대 한 그들의 접근에서 그들의 관계 되는 풍부 때문에 치료 잠재력을 전시 하는 건의 했다. 따라서, ASCs 치료 용 줄기 세포의 좋은 원천이 될 나타납니다. 이 연구에서는 일반 루이스 쥐의 epididymal 지방 지방에서 ASC 시트 온도 응답 문화 요리와 의약품을 포함 하는 일반 문화 매체를 사용 하 여 성공적으로 창조 되었다. ASC 시트 커 당뇨병 지방산 (ZDF) 쥐에 이식 했다의 저하는 비만 2 형 당뇨병 쥐 모델 상처 치유. 상처 후부 두개골 표면에 만들어진, ASC 시트는 상처에 이식 했다 그리고 bilayer 인공 피부는 시트 커버를 사용 되었다. 받은 ASC 시트에서 쥐에서 쥐 ASC 시트 이식 없이 보다 치유 상처 더 했다. ASC 시트는 습 한 상처 환경 유지 보수를 필요로 건조 조건에 과민 하기 때문에이 접근은 제한 되었다. 따라서, 인공 피부 건조를 방지 하는 ASC 시트 커버를 사용 되었다. 인공 피부와 함께에서 ASC 시트 allogenic 이식 또한 다른 다루기 힘든 궤 양이나 화상, 말 초 동맥 질환 및 교원 질 질병, 관찰 등을 적용할 수 있습니다 그리고는 영양 또는 스테로이드를 사용 하는 환자에 게 관리 될 수 있습니다. 따라서,이 치료는 당뇨병 상처 치유에 대 한 치료 옵션을 개선 향한 첫 번째 단계를 수 있습니다.

이 프로토콜 다루기 힘든 당뇨병 상처에 대 한 새로운 세포 기반 치료를 설정 하려고 했습니다. ( 그림 1에서 그림)로 짧게 수용자 rASC 시트 셀 시트 엔지니어링을 사용 하 여 정상적인 쥐에서 만들어진 하 고 전체 간격 피부 결함에 인공 피부의 bilayer를 사용 하 여 당뇨병 쥐에 이식 했다. 가벼운 현미경의 이미지 rASC 시트 (그림 2A)의 좋은 본보기와 ...

Watch this Scientific Journal Video about Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model at JoVE.com

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

지방 유래 줄기 세포 (ASCs) 쉽게 격리 하 고 정상 쥐의 지방에서 수확. ASC 시트 셀 시트 엔지니어링을 사용 하 여 만들 수 있습니다 하 고 주 커 전체 두께 전시 하는 당뇨 지방 쥐 결함 노출 된 뼈와 피부와 다음 인공 피부의 bilayer 덮여에 이식 될 수 있다.

생성 및 당뇨병 치유 하는 부상을 모델에서 줄기 세포 지방이 많은 파생 (ASC) 시트의 이식

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

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Research

JoVE Journal

Medicine

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

11.3K 조회수.  Tokyo Women's Medical University School of Medicine. 지방 유래 줄기 세포 (ASCs) 쉽게 격리 하 고 정상 쥐의 지방에서 수확. ASC 시트 셀 시트 엔지니어링을 사용 하 여 만들 수 있습니다 하 고 주 커 전체 두께 전시 하는 당뇨 지방 쥐 결함 노출 된 뼈와 피부와 다음 인공 피부의 bilayer 덮여에 이식 될 수 있다.

비디오: Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

허혈성 상처 모델 생산을위한 단순화된 테크닉

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

연구

의학

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

당뇨병 쥐의 부고환 지방 세포에 이식 된 지지체 지지대 지지대

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

생체공학

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3 차원 동적 문화 시스템과 다층된 중간 엽 줄기 세포 시트의 건설

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The development of diversified technologies combining adipose tissue-derived stromal cells (ADSCs) with various biomaterials has led to the construction of numerous types of tissue-engineered substitutes, such as bone, cartilage, and adipose tissues from rodent, porcine, or human ADSCs. Extended esophageal endoscopic submucosal dissection (ESD) is responsible for esophageal stricture formation. Stricture prevention remains challenging, with no efficient treatments available. Previous studies reported the effectiveness of mucosal cell-sheet transplantation in a canine model and in humans. ADSCs are attributed anti-inflammatory properties, local immune modulating effects, neovascularization induction, and differentiation abilities into mesenchymal and non-mesenchymal lineages. This original study describes the endoscopic transplantation of an ADSC tissue-engineered construct to prevent esophageal stricture in a swine model. The ADSC construct was composed of two allogenic ADSC sheets layered upon each other on a paper support membrane. The ADSCs were labeled with the PKH67 fluorophore to allow probe-based confocal laser endomicroscopy (pCLE) monitoring. On the day of transplantation, a 5-cm and hemi-circumferential ESD known to induce esophageal stricture was performed. Animals were immediately endoscopically transplanted with 4 ADSC constructs. The complete adhesion of the ADSC constructs was obtained after 10 min of gentle application. Animals were sacrificed on day 28. All animals were successfully transplanted. Transplantation was confirmed on day 3 with a positive pCLE evaluation. Compared to transplanted animals, control animals developed severe strictures, with major fibrotic tissue development, more frequent alimentary trouble, and reduced weight gain. In our model, the transplantation of allogenic ADSCs, organized in double cell sheets, after extended ESD was successful and strongly associated with a lower esophageal stricture rate.

This study reports a successful method of endoscopical adipose tissue-derived stromal cell (ADSC)-sheet transplantation for esophageal stricture prevention after an extended endoscopic submucosal dissection (ESD) in a swine model.

ADSC 매 이식은 확장 식도 내시경 점막하 절개 후 협착을 방지하는

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

쥐 모델에 부분 Colectomy 후 누설을 줄이기 위해 지방 조직 유래 줄기 세포의 이식

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

당뇨병 마우스에서 만성 상처를 만드는 프로토콜

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Prospective, Randomized, and Controlled Study of a Human Umbilical Cord Mesenchymal Stem Cell Injection for Treating Diabetic Foot Ulcers

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with dressing changes, hyperbaric oxygen, and vacuum sealing drainage are the main conservative treatments in clinical practice, and large wounds often require skin grafts or skin flap grafts. However, the treatment effects are not ideal, and many complications exist. Due to its complex pathogenesis, long treatment time, significant associated difficulties, and high disability rate, diabetic foot ulcers cause a heavy burden to patients, society, and medical care. According to our previous study, the pharmacological effects of human umbilical cord blood stem cells include nonspecific immune regulation; increased secretion of growth factors, vasoactive factors, and anti-inflammatory factors; enhanced anti-infectious ability of the human body; elimination of inflammation; and promotion of angiogenesis and ulcer healing. These effects suggest stem cells may be useful as an autologous or allogeneic treatment for refractory wounds. Therefore, we are conducting a clinical trial to treat refractory diabetic wounds with human umbilical cord stem cells in our clinic for diabetic foot ulcer patients who meet the inclusion criteria.

The present protocol describes a prospective, randomized, controlled clinical study that evaluates a human umbilical cord mesenchymal stem cell injection for the treatment of chronic diabetic foot ulcers.

당뇨병성 족부궤양 치료를 위한 인간 탯줄 중간엽 줄기세포 주사의 전향적, 무작위 및 대조 연구

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

뮤린 아일렛의 인과 피하 백색 지방 조직 (ISWAT) 이식 모형

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

This study presents a protocol for fabricating core-sheath 3D-bio-printed scaffolds for chronic wound healing. Extracellular vesicles are isolated from mesenchymal stem cells, and loaded into the core (alginate) with the sheath made from carboxymethyl cellulose and alginate lyase. This design allows controlled scaffold degradation and efficient EVs release.

만성 상처 관리를 위한 Core-sheath 3D-Bioprinted Scaffold를 사용한 세포외 소포 전달 최적화

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol ...

A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal models of type 1 diabetic wounds. However, improper manipulation can lead to model instability and high mortality in rats. Unfortunately, there are few existing guidelines on type 1 diabetic wound modeling, and they lack detail and do not present specific reference strategies. Therefore, this protocol details the complete procedure for constructing a type 1 diabetic wound model and analyzes the progression and angiogenic characteristics of the diabetic wounds. Type 1 diabetic wound modeling involves the following steps: preparation of the streptozotocin injection, induction of type 1 diabetes mellitus, and construction of the wound model. The wound area was measured on day 7 and day 14 after wounding, and the skin tissues of the rats were extracted for histopathological and immunofluorescence analysis. The results revealed that type 1 diabetes mellitus induced by 55 mg/kg streptozotocin was associated with lower mortality and a high success rate. The blood glucose levels were relatively stable after 5 weeks of induction. The diabetic wound healing rate was significantly lower than that of normal wounds on day 7 and day 14 (p &#60; 0.05), but both could reach more than 90% on day 14. Compared with the normal group, the epidermal layer closure of diabetic wounds on day 14 was incomplete and had delayed re-epithelialization and significantly lower angiogenesis (p &#60; 0.01). The type 1 diabetic wound model constructed based on this protocol has the characteristics of chronic wound healing, including poor closure, delayed re-epithelialization, and decreased angiogenesis compared to normal rat wounds.

The streptozotocin-induced diabetic wound model in male SD rats is currently the most widely used model for studying wound healing in type I diabetes mellitus. This protocol describes the methods used to construct this model. It also presents and addresses potential challenges and examines the progression and angiogenic characteristics of diabetic wounds.

제1형 당뇨병의 쥐 상처 모델을 구축하기 위한 프로토콜

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal ...