著者は、実用的なアドバイスを提供するため医学部形成外科、再建外科、順天堂大学医学部の博士占部をありがとうございます。糖尿病センターの東京女子医科大学医学部優れたテクニカル サポートのための村田英一氏も感謝いたします。本研究は、教育省、文化、スポーツ、科学、技術 (文部科学省) 日本から開発途上技術革新システム「細胞シート組織工学センター (CSTEC)」のプロジェクトの学際的研究領域プログラムを高度なイノベーション センターの作成によって支えられました。

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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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+effect+of+adipose-derived+stem+cells:+a+critical+role+of+secretory+factors+on+human+dermal+fibroblasts.">Wound healing effect of adipose-derived stem cells: a critical role of secretory factors on human dermal fibroblasts.</a> J Dermatol Sci. 48 (1), 15-24 (2007).</li><li>Nakagami, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+autologous+cell+therapy+in+ischemic+limb+disease+through+growth+factor+secretion+by+cultured+adipose+tissue-derived+stromal+cells.">Novel autologous cell therapy in ischemic limb disease through growth factor secretion by cultured adipose tissue-derived stromal cells.</a> Arterioscler Thromb Vasc Biol. 25 (12), 2542-2547 (2005).</li><li>Asahara, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+contributes+to+postnatal+neovascularization+by+mobilizing+bone+marrow-derived+endothelial+progenitor+cells.">VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.</a> EMBO J. 18 (14), 3964-3972 (1999).</li><li>Kato, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+transplantation+of+an+adipose-derived+stem+cell+(ASC)+sheet+combined+with+artificial+skin+accelerates+wound+healing+in+a+rat+wound+model+of+type+2+diabetes+and+obesity.">Allogeneic transplantation of an adipose-derived stem cell (ASC) sheet combined with artificial skin accelerates wound healing in a rat wound model of type 2 diabetes and obesity.</a> Diabetes. , db141133 (2015).</li></ol>

次の著者は、このパブリケーションに関連する金融関係を開示: 岡野光夫氏が創設者であり、技術と東京女子医科大学からの特許のライセンスを取得、セル ・ シード社理事長、岡野光夫・大和雅之がセル シード株式会社東京女子医科大学の関係者がセル ・ シード社から研究資金を受け取る他の著者は、この文書に関連する金融関係を持たないを宣言します。

RASC シートを正常に培養するための最も重要な手順は次のとおりです: 1) 温度は、温度応答性培養皿上での培養中におよそ 37 ° C に保たれなければなりません。RASC シートの作成時にすべてのプロシージャを 37 ° C の熱板に行ったし、すべての試薬は自発的に料理31から切り離す細胞を防ぐために 37 ° C に加温しました。2) 受信者 ZDF ラットを監視すると、rASC シートの巧妙な?...

糖尿病患者の人口は世界的に増加しており 4 億 2015年1;糖尿病患者の約 15-25% は、下肢糖尿病性潰瘍2の進行からのリスクです。下肢糖尿病性潰瘍は難治性で、完全な回復後のリハビリ訓練の長期治療を必要とする可能性があります。頻繁に長い治療期間は、患者の生活の質の大幅な削減の結果します。したがって、糖尿病の傷の治療のために減らすか、悪化を防ぐ新たな治療法が開発されなければなりません。糖尿病創傷治癒過程を評価するため我々 は、糖尿病性潰瘍創傷治癒モデル ラットでは, 実用的な臨床的条件を模倣する、最適化し、創傷治癒促進細胞シート工学を用いた脂肪由来幹細胞 (ASC) シートを移植かどうか評価します。
間葉系間質細胞 (MSCs) は、様々 な細胞の血統3に分化する能力を彼らの免疫調節効果、自己再生能力のための創傷治癒を促進するための優れたポテンシャルを展示します。MSCs パラクリン活動4、5潜在的な血管新生を含む他のティッシュから得られる利点を展示して Asc が脂肪組織由来 MSC の一種。脂肪組織は人間の体内では、比較的豊富なでき、アクセシビリティ、低侵襲の手順を使用して、コレクションです。したがって、Asc は創傷治癒のアプリケーション6、7の実験に使用されています。
以前のレポートは、傷の周りの地域に単一細胞 MSC 懸濁液の直接注入が創傷治癒8,9を加速することが示されています。ただし、単一細胞懸濁液を注入糖尿病性潰瘍モデルにおける創傷治癒の加速のレポート、にもかかわらず、創傷部位に移植細胞の生存時間は明確ではないです。
本研究では、温度応答性培養皿を用いた細胞シート工学を適用されます。これらの料理はある温度応答性高分子Nイソプロピルアクリルアミド彼らの表面の10に共有結合です。グラフト高分子層温度制御細胞接着や培養皿の表面から剥離が可能です。皿の表面は 37 ° c、それは 32 ° C 以下の温度では親水性になったとき、細胞が自発的に表面から切り離すに対し、細胞が接着し、増殖を許可する疎水性になります。の温度を減らすことによって単にそのまま細胞間結合と細胞外マトリックス (Ecm) の連続した細胞シートとして培養細胞を収穫できます。したがって、損傷、トリプシンなどの ECM 蛋白質分解酵素は必要な11ではありません。したがって、細胞シート工学は、細胞間の接続を維持でき、細胞移植の有効性を改善することができます。
さらに、細胞シート移植細胞注入12と比較した場合の細胞生存率が増加します。このプロトコル Zucker 糖尿病脂肪 (ZDF) ラットは、創治癒遅延と 2 型糖尿病や肥満モデルとして選ばれました。ZDF ラットは、自発的に約 4 週間で肥満を開発します。彼らは、8、12 週齢、その時点で発揮する高血糖インスリン抵抗性、脂質異常症、高トリグリセリド血症13に関連付けられている間肥満と 2 型糖尿病を開発します。創傷治癒、血流の低下、末梢血管、糖尿病性腎症14,,1516観察も。また、ZDF ラット糖尿病性潰瘍などの難治性皮膚潰瘍の治癒過程を研究するための適切なモデルがあります。
人間および齧歯動物の創傷治癒のメカニズムの違いは、皮膚の解剖学的差異に関連付けられます。正常ラットの創傷治癒は、再上皮化および肉芽組織の形成に基づいて人間の創傷治癒過程に対し創収縮に基づいています。通常、傷副木齧歯動物モデルで使用される創収縮を最小限に抑え、肉芽組織17、漸進的な形成を可能が非糖尿病ラットで傷はほぼ完全に収縮によって閉じられます。ただし、糖尿病創傷収縮 ZDF ラットは障害者と創傷治癒の主が再上皮化および肉芽組織形成を介して行われますしたがって、このプロセスより人間の創傷治癒の14に似ています。
糖尿病性創傷デブリドマンが臨床的によく遭遇後露出した骨付き。無胸腺ヌードマウス18,19の背中に直径 12 mm 全厚皮傷や正常マウス20の背中に直径 10 mm 全厚皮の傷、前が調査されました。重度の糖尿病の傷のための臨床モデルを開発するには、より大きい (15 × 10 mm2) 全層皮膚欠陥は骨を露出し、骨膜なしとして作成された、2 型糖尿病と肥満ラットで、前に説明した21。
通常の Lewis ラットの Asc からラット ASC (rASC) シートは、ASC シートの同種移植によって作成されました。臨床実習、潰瘍糖尿病患者は多くの場合、制御不能な高血糖と高体重指数、これら合併症原因創傷治癒障害これらの患者からの脂肪組織の取得の難易度を高めるなど、重度の糖尿病合併症を示すために、自家移植が可能。さらに、糖尿病を展示動物から Asc プロパティを変更、関数22障害者します。したがって、ここで示されるプロトコルは、正常ラットから rASC シートの同種移植と人工皮膚の糖尿病ラットへの応用について説明します。
このプロトコルで使われる膜人工皮膚の傷の自発収縮を防ぐことができます、新しい結合組織のマトリックスの合成を促進して true 真皮23を似ています。このプロトコルでは、人工皮膚は rASC シート上に配置、創収縮または緩いラット皮膚から結果の拡大を防ぐために糸で固定します。さらに、人工皮膚が移植された ASC シートや傷の湿潤環境を維持、感染や外部の力から傷を保護に ASC シート三次元フレームワークを提供します。最後に、非粘着ドレッシングは、外的な影響から保護、湿潤環境の維持、滲出液を吸収する創傷で配置されます。
RASC シートは薄い、柔軟性、および変形を暴行心24など、受信者のサイトに移動に付着することができます。細胞シート工学は様々 な組織の再建のために使用されているし、治療効果25,26.を生成することができます。臨床治療の可能性を示す ASC シートは、多くのタイプの傷の治癒を促進可能性があります。さらに、ASC シート、人工皮膚の使用と組み合わせるの同種移植は難治性の潰瘍や末梢動脈疾患や膠原病にみられるなど、火傷の治療に適用されるかもしれないまたは彼らは栄養不足ですか、ステロイドを使用している患者に投与されることがあります。このアプローチには、Asc を移植の効率が上がります。ZDF ラットの創傷治癒モデル人間の創傷治癒過程に似ており、小型の実験動物の臨床条件を模倣する重度の巻き具合が生成されます。

<table border="0" cellpadding="0" cellspacing="0" width="678">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">&#945;-MEM glutamax</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:119px;">32571-036</td>
			<td style="width:167px;">Carlsbad, CA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Japan Bioserum Co Ltd.</td>
			<td style="width:119px;">S1650-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">10 103 578 001</td>
			<td>Mannheim, Germany</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">60-cm2 Primaria tissue culture dish</td>
			<td>BD Biosciences</td>
			<td>353803</td>
			<td>Franklin Lakes, NJ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco&#39;s Phosphate Buffer Saline (PBS)</td>
			<td>Life Technologies</td>
			<td style="width:119px;">1490-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-ethylenediamine tetraacetic acid (EDTA)</td>
			<td>Life Technologies</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-ascorbic acid phosphate magnesium salt n-hydrate</td>
			<td style="width:169px;">Wako</td>
			<td>013-19641</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">35-mm temperature-responsive culture dish (UpcellTM)</td>
			<td>CellSeed</td>
			<td>NUNC-174904</td>
			<td>Tokyo, Japan</td>
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		<tr height="42">
			<td height="42" style="height:42px;">Microwarm plate (MP-1000)</td>
			<td style="width:169px;">Kitazato Science Co., Ltd.</td>
			<td style="width:119px;">1111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rodent mechanical ventilator</td>
			<td>Stoelting</td>
			<td>#50206</td>
			<td>Wood Dale, IL</td>
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		<tr height="21">
			<td height="21" style="height:21px;">4% isoflurane</td>
			<td>Pfizer Japan</td>
			<td>114-13340-3</td>
			<td>Tokyo, Japan</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Artificial skin (Pelnac&#174;)</td>
			<td>Smith &#38; Nephew</td>
			<td>PN-R40060</td>
			<td>&#160;Tokyo, Japan</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Non-adhesive dressing (Hydrosite plus&#174;)</td>
			<td>Smith &#38; Nephew</td>
			<td align="right">66800679</td>
			<td>Known as Allevyn non-adhessing&#174; in the United State</td>
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		<tr height="42">
			<td height="42" style="height:42px;">5-0 nylon suture</td>
			<td>Alfresa</td>
			<td style="width:119px;">EP1105NB45-KF2</td>
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		<tr height="21">
			<td height="21" style="height:21px;">20 CELLSTAR TUBES</td>
			<td>greiner bio-one</td>
			<td style="width:119px;">227 261</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:223px;">15mL Centrifuge Tube</td>
			<td>Corning Incorporated</td>
			<td align="right" style="width:119px;">430791</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:223px;">14 GOLDMAN-FOX PERIOSTEAL</td>
			<td>Hu-Friedy</td>
			<td style="width:119px;">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

人工皮膚は、臨床実習でかなり治療成績を成し遂げた。ただし、妨げられる血流や大きな傷、糖尿病患者の傷のため人工皮膚の治療が長引く可能性も。細胞ベースの治療は糖尿病性の潰瘍の治療のための新しい手法として登場している、細胞シート工学は、細胞移植の有効性を改善しました。レポートの数は、脂肪由来幹細胞 (Asc)、間葉系間質細胞 (MSC) の種類は、脂肪組織と他の組織から MSCs と比較されたときコレクションのユーザー補助機能の相対的な豊かさのための治療の可能性を示すことを示唆しています。したがって、Asc は、幹細胞治療上の使用のための良いソースに表示されます。本研究では通常の Lewis ラットの副睾丸脂肪脂肪から ASC シートが作成されましたとアスコルビン酸を含む通常の培養液に温度応答性培養皿を用いたします。ASC シート Zucker 糖尿病性脂肪酸 (ZDF) ラットに移植、2 型糖尿病の減少を示す肥満モデルラット創傷治癒します。傷は後部の頭蓋表面上に作成された、ASC シートが傷に移植した膜人工皮膚シートをカバーするため使用されました。ASC シートを受け取った ZDF ラット ASC シートの移植なし ZDF ラットよりも癒しの傷があった良い。このアプローチは、ASC シート湿潤環境の維持を必要とする、乾燥した条件に敏感であるために限られていた。したがって、人工皮膚は、乾燥を防ぐために ASC シートをカバーしていました。人工皮膚との組み合わせで ASC シートの同種移植も他の難治性潰瘍や火傷、末梢動脈疾患と膠原病、観察などに適用して栄養不足ですか、ステロイドを使用している患者に投与されることがあります。したがって、この治療は糖尿病創傷治癒の治療方法の改善への第一歩かもしれません。

このプロトコルでは、セルベースの難治性の糖尿病性創傷の治療法を確立しようとしています。(図 1に示す) として簡潔に同種 rASC のシーツは細胞シート工学を用いた正常ラットから作成された、糖尿病ラットの重層全層皮膚欠損創に人工皮膚を使用してそれから移植します。RASC シート (図 2 a) の良い例と悪い例 rASC シート (図 2 b</stron...

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

脂肪由来幹細胞 (Asc) は簡単に隔離され、正常ラットの脂肪から採取しました。ASC のシーツは、細胞シート工学を使用して作成することができます、ザッカー出展全層脂肪の糖尿病のラット皮膚の露出した骨の欠陥と人工皮膚の層で覆われてに移植することができます。

作成および糖尿病創傷治癒モデルにおける脂肪由来幹細胞 (ASC) シートの移植

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

creation-transplantation-an-adipose-derived-stem-cell-asc-sheet

Research

JoVE Journal

Medicine

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

11.3K ビュー.  Tokyo Women's Medical University School of Medicine. 脂肪由来幹細胞 (Asc) は簡単に隔離され、正常ラットの脂肪から採取しました。ASC のシーツは、細胞シート工学を使用して作成することができます、ザッカー出展全層脂肪の糖尿病のラット皮膚の露出した骨の欠陥と人工皮膚の層で覆われてに移植することができます。

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

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

同所性大動脈移植：慢性血管障害の発生研究のラットモデル

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

医学

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

のマウスモデル子宮内で移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

における幹細胞移植 in vitroで模擬虚血/再灌流モデル

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

創傷治癒のマウスモデル

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

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.

ラットモデルにおける部分的な術後後の漏れを減らすために脂肪組織由来幹細胞シートの移植

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

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

前のヴィヴォ角膜器官培養モデルの創傷治癒の研究

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

ターゲット灰白交通枝選択的化学腰椎交感神経切除術で

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

増殖糖尿病網膜症における線合併症の体外培養モデル

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

作成および糖尿病創傷治癒モデルにおける脂肪由来幹細胞 (ASC) シートの移植

要約

さらに動画を探す

この動画の章

同所性大動脈移植：慢性血管障害の発生研究のラットモデル

のマウスモデル子宮内で移植

における幹細胞移植 in vitroで模擬虚血/再灌流モデル

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を決定するために、定量的リアルタイムPCRを用いて

創傷治癒のマウスモデル

ラットモデルにおける部分的な術後後の漏れを減らすために脂肪組織由来幹細胞シートの移植

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

前のヴィヴォ角膜器官培養モデルの創傷治癒の研究

ターゲット灰白交通枝選択的化学腰椎交感神経切除術で

増殖糖尿病網膜症における線合併症の体外培養モデル

作成および糖尿病創傷治癒モデルにおける脂肪由来幹細胞 (ASC) シートの移植

要約

さらに動画を探す

この動画の章

同所性大動脈移植：慢性血管障害の発生研究のラットモデル

のマウスモデル子宮内で移植

における幹細胞移植 in vitroで模擬虚血/再灌流モデル

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

創傷治癒のマウスモデル

ラットモデルにおける部分的な術後後の漏れを減らすために脂肪組織由来幹細胞シートの移植

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

前のヴィヴォ角膜器官培養モデルの創傷治癒の研究

ターゲット灰白交通枝選択的化学腰椎交感神経切除術で

増殖糖尿病網膜症における線合併症の体外培養モデル

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を決定するために、定量的リアルタイムPCRを用いて