We gratefully acknowledge the financial support provided by the Next-Generation BioGreen 21 Program (No. PJ007969), Rural Development Administration, and the National Research Foundation (grant no. NRF-2015R1D1A1A01056639) of the Republic of Korea.

動物実験は、慶尚大学校での生物医学実験用動物センターによって承認されました。 動物手順1.準備<ol><li>骨髄および滑液からのMSCの非侵襲的なコレクションのために成人女性のミニブタを準備します。麻酔および試料収集の1日前ミニブタの臨床検査を実行します。 <ol><li>完全血球計数検査を使用して、体温、呼吸数、および心拍数を含む健康診断のための、および豚の行動と血液学的状態の観察のために臨床的に正常な動物を提供します。 注：臨床的に健康なミニブタのパラメータ間隔は11です - 呼吸数29 /分、68 - 心拍数98 /分、および37 - 体温のために38°C。 </li></ol></li><li> 12時間のPRIO - 6の術前絶食期間中にケージからすべての食用の寝具を削除若者や成人のミニブタのための麻酔の投与rは、空腹時の3時間は、新生児のために観察されるはずです。麻酔時まで飲料水を提供します。 </li></ol>麻酔のための動物の作製<ol><li>アセプロマジン（0.1ミリグラム/キログラム）、メデトミジン（0.03ミリグラム/ kg）で麻酔の投与前ミニブタを薬で治療し、アトロピン（0.04ミリグラム/キログラム）。 <ol><li>成人ミニブタについて少なくとも30mmの長さの21 G針を使用して、すべての薬剤を筋肉内（IM）を注射します。横子宮頸部筋肉内または大腿部筋肉に1〜3センチメートル耳の後ろにIM注射を実行します。 注意：動物で恐怖やストレスを誘発または安全に前投薬​​を注入するために乱暴な取り扱いを必要とせずに、すべての手順を実行します。 </li></ol></li><li>背臥位でミニブタを置きます。 </li><li> 15分前投薬の投与後、1.5％イソフルラン管理を使用して麻酔を開始吸入を介してistered。 <ol><li>キャリアガスとして酸素（/分1.5 L）で、2.5％の濃度の増加に続いて、気管内チューブを介してイソフルランの吸入（1.5％）を継続します。 </li></ol></li><li>連続麻酔中ミニブタの心拍数、体温、および経皮的血液酸素飽和度（SpO 2）モニタ。体温を維持するために39°C - 38の温度で循環水ブランケットを使用してください。麻酔中に乾燥から目を保護するために眼科軟膏を適用します。 </li><li>麻酔13の深さを評価するGuedelの分類に従ってください。 </li></ol>細胞の単離および培養のための3の準備<ol><li> 10％ウシ胎児血清（FBS）を補充した高度ダルベッコ改変イーグル培地（ADMEM）、1％グルタマックス500mlの、塩基性線維芽細胞成長ファクトを10ng / mlのを準備する、骨髄および滑液由来するMSCを培養しますR（bFGFを）、および1.0％ペニシリン - ストレプトマイシン（それぞれ万IU 10,000μgの/ mlの、ペニシリン - ストレプトマイシン）。 <ol><li> CO 2インキュベーター内で5％CO 2の加湿雰囲気中で38.5℃でADMEMをインキュベートします。 </li></ol></li><li>細胞分離および洗浄のための製造業者の指示に従ってダルベッコのリン酸緩衝生理食塩水（DPBS）500mlのを準備します。 </li></ol> 4.ミニブタから骨髄を収集<ol><li>エリア腸骨稜（ 図1）上のサイズは約15×15センチ選択し、滅菌剃刀や電気バリカンを使用して、この領域から毛を取り除きます。ポビドンヨードおよび70％エタノール：ミニブタが配置されると、交互に次の消毒剤を使用して手順サイトを3回スクラブ。領域が完全に乾燥した後、滅菌ドレープを適用します。 </li><li> ejec前にヘパリンの4ミリリットル - プレコート10mlを3でそれをすすぐことによって、ヘパリンの100 U / mlのシリンジティン抗凝固剤。 注：骨髄穿刺時にシリンジ内の凝固を避けるために、この手順を実行します。 </li><li>しっかりとヘパリンプレコートされた10ミリリットルの注射器に取り付けられた骨髄抽出（3.0×100ミリメートル）を使用して、麻酔下でミニブタの腸骨稜骨から骨髄の5ミリリットルの最小値を抽出します。 </li></ol>骨髄からとインビトロ栽培で抽出した細胞の単離5. <ol><li>抽出された骨髄から細胞を単離します。 <ol><li>室温で15 mLのコニカルチューブ中のDPBSの等量で抽出した骨髄を希釈します。 </li><li> 15ミリリットルコニカルチューブに密度遠心分離培地（フィコール・パック）の3ミリリットルを追加します。 </li><li>室温で40分間、400×gでで4 mLの混合せずに密度遠心分離媒体上に配置されるDPBSで骨髄の層、及び遠心分離して希釈します。 </li><li>単核細胞層を回収します血漿（上層）と密度遠心分離媒体（下層）を含む二つの層の間に軟膜から（多国籍企業）、そして15mlコニカルチューブに多国籍企業を移します。 </li><li> 7で転送多国籍企業を希釈 - DPBSの8ミリリットル、室温で10分間、500×gで、それらを遠心分離します。上清を捨て、DPBSで細胞を2回洗浄します。 </li><li> ADMEM 2ml中の細胞ペレットを中断し、35mm組織培養皿に移します。 5％CO 2の加湿雰囲気中で38.5℃で培養皿をインキュベートします。 </li></ol></li><li>骨髄から単離された細胞のインビトロ培養で 。 <ol><li> 48時間後、皿に付着していない細胞を含む培地を吸引し、ADMEMの2ミリリットルを交換し、5％のCO 2の加湿雰囲気中で38.5℃で静置します。すべての第三日間培養液を交換します。 </li><li> 80％の密集度、DPBSで細胞を洗浄し、0.25％の1mlでそれらを分離 - 70でトリプシン/ EDTA、3 37℃でそれらをインキュベートする - 細胞が剥がれるまで5分。 </li><li> ADMEMを10mlを用いて細胞を収穫を15mlコニカルチューブにそれらを転送し、室温で5分間、300×gで、それらを遠心。上清を捨てます。 </li><li>新鮮なADMEMの1ミリリットルで収穫された細胞を懸濁し、血球計数器を使用してそれらを数えます。 </li><li> 5に細胞を置き- ADMEMの4ミリリットルを含む7.5×10 5 / 25Tフラスコ、5％CO 2の加湿雰囲気中で38.5℃でそれらをインキュベートします。二日おきに培地を変更します。 </li></ol></li></ol>ミニブタから関節液の6集<ol><li>骨髄穿刺するための手順と同様に、大腿関節の上に約15×10センチメートル（長さ/幅）の適切な領域を選択し、滅菌剃刀や電気バリカンを使って髪を除去、消毒手順（ステップ4.1）によって従ってください。 </li><li> 23 Gで3 mlシリンジを挿入触診のランドマーク（膝蓋骨と脛骨稜）の後に関節に針。 <ol><li>関節における滑液の吸引のための注射器に穏やかな吸引を適用します。 1ミリリットルが、これはミニブタや関節の大きさに応じて変化する - 関節から採取した滑液のおおよその体積は0.5です。滑液の収集量が少なすぎると滑液腔にDPBSを通してフラッシュします。 </li><li>直ちに血液による汚染を避けるために注射器を引き抜きます。 </li></ol></li></ol>収集された関節液からおよびインビトロ栽培7.細胞の単離<ol><li>吸引滑液から細胞を分離します。 <ol><li>室温で15ミリリットルコニカルチューブ内のDPBS等量の吸引滑液を希釈します。 </li><li>破片を除去するために、40μmのナイロン細胞ストレーナーを用いて希釈滑液をフィルタリングします。 </li><li>フィルタリング滑膜を含むチューブにDPBSの10ミリリットルを追加します。流体、室温で10分間、400×gで遠心分離して。上清を捨て、DPBSで細胞を2回洗浄します。 </li><li> 2のセルを配置し、ADMEM 2ml中の細胞ペレットを懸濁- ADMEM 2mlで3×10 5細胞/ 35 mm組織培養皿、および5％CO 2の加湿雰囲気中で38.5℃でそれらをインキュベート。 </li></ol></li><li>孤立した滑液由来MSCのin vitro培養のための手順を繰り返し5.2。 </li></ol>ミニブタの8.ポスト手順ケア<ol><li>必要に応じて、骨髄採取後、皮膚ステープラーを使用して、単一の縫合糸を用いて骨髄抽出注射部位の皮膚を閉じます。 </li><li>手順が完了した後、3日間1日1回ミニブタへのエンロフロキサシン（5mg / kg）およびメロキシカム（0.2ミリグラム/ kg）を筋肉内注射を投与します。 </li><li>手順が完了した後、骨髄および滑液収集部位を消毒3日間、一日一回、0.1％ポビドンヨードを持ちます。慎重な合併症の兆候（ 例えば 、腫れ、膿、または赤み）を監視するために毎日観察します。 </li></ol>フローサイトメトリーを使用した9セルの表現型<ol><li>継代3 80％コンフルエント - 5 - 、DPBSで細胞を洗浄し、0.25％（v / v）のトリプシン/ EDTA 1mlで扱うMSCは70である場合。細胞が剥離するまで5分 - そして、3 37℃でそれらをインキュベートします。 </li><li> DPBSを10mlを用いて細胞を収穫を15mlコニカルチューブにそれらを転送し、室温で5分間、300×gで、それらを遠心。上澄み液を捨てます。 </li><li>細胞を固定し、3.7％ホルムアルデヒド溶液10mlに細胞ペレットを懸濁し、分析前に一日4℃でそれらを維持します。 <ol><li>ある日固定した後、DPBSは二回5分毎時間、300×gで遠心分離を用いて細胞を洗浄。上澄み液を捨てます。 </li><li> 4ミリリットル中に細胞ペレットを一時停止1％（w / v）のウシ血清アルブミン（BSA）を補充し、非特異的Fc媒介性相互作用をブロックするために4℃で1時間ペレットをインキュベートさDPBSの。 </li><li> 1.5ミリリットルチューブに細胞懸濁液の500μlのアリコートを配布します。 </li><li>非結合抗体（CD29及びビメンチン）のために、3分間300×gでの遠心分離によって細胞を洗浄します。上澄み液を捨てます。 </li><li> 1500μlの中で細胞ペレットサスペンド：一次抗体の100倍希釈をし、4℃で1時間静置します。 </li><li>毎回5分間、300×gで遠心分離することにより、1％ウシ血清アルブミンで細胞を2回洗浄します。上澄み液を捨てます。 </li><li>二次抗体を抱合フルオレセインイソチオシアネート（FITC）の100倍希釈し、4℃の暗所で1時間のためにそれをインキュベート：1500μlの中で細胞ペレットを中断します。 </li></ol></li><li> FITC結合抗体（アイソタイプ、CD34、CD44およびCD45）について、centrifuによって細胞を洗浄3分間300×gでゲーション。上澄み液を捨てます。 <ol><li> 1500μlの中で細胞ペレットサスペンド：100 FITC結合抗体を、4℃の暗所で1時間のためにそれをインキュベートします。 </li><li>両方の染色条件のために、5分間G×300での遠心分離によってDPBSで2回時間細胞を洗浄します。上澄み液を捨てます。 </li><li> DPBS500μlの中に細胞をサスペンド、5ミリリットル丸底チューブにそれらを転送し、8フローサイトメーターを使用して結果を分析します。 </li></ol></li></ol>

<ol>
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W., Niemann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+perspectives+for+porcine-to-human+xenografts.">The perspectives for porcine-to-human xenografts.</a> Comp Immunol Micro biol Infect Dis. 32 (2), 91-105 (2009).</li><li>Zeng, L., 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=Multipotent+adult+progenitor+cells+from+swine+bone+marrow.">Multipotent adult progenitor cells from swine bone marrow.</a> Stem Cells. 24 (11), 2355-2366 (2006).</li><li>Zeng, L., 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=Bioenergetic+and+functional+consequences+of+bone+marrow-derived+multipotent+progenitor+cell+transplantation+in+hearts+with+postinfarction+left+ventricular+remodeling.">Bioenergetic and functional consequences of bone marrow-derived multipotent progenitor cell transplantation in hearts with postinfarction left ventricular remodeling.</a> Circulation. 115 (14), 1866-1875 (2007).</li><li>Sekiya, I., 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=Human+mesenchymal+stem+cells+in+synovial+fluid+increase+in+the+knee+with+degenerated+cartilage+and+osteoarthritis.">Human mesenchymal stem cells in synovial fluid increase in the knee with degenerated cartilage and osteoarthritis.</a> J Orthop Res. 30 (6), 943-949 (2012).</li><li>Mochizuki, 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=Higher+chondrogenic+potential+of+fibrous+synovium-+and+adipose+synovium-derived+cells+compared+with+subcutaneous+fat-derived+cells:+Distinguishing+properties+of+mesenchymal+stem+cells+in+humans.">Higher chondrogenic potential of fibrous synovium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: Distinguishing properties of mesenchymal stem cells in humans.</a> Arthritis Rheum. 54 (3), 843-853 (2006).</li></ol>

The authors declare that they have no conflict of interest.

ミニブタは、骨髄および滑液からのMSCの単離を確立するために使用しました。年齢、性別、および疾患などの様々な生理的条件を、排除するために、同質遺伝子背景ドナーからのミニブタは正確に細胞源に依存する特性化を評価するために選ばれました。ブタであることが知られている解剖学的、生理学的、および遺伝的にヒトに類似し、特に、サイズが一致した臓器を作り出すことができ?...

Multipotent mesenchymal stem cells (MSCs) can be classified into mesenchymal cell lineages, and MSCs have been established and isolated from various tissue sources, such as from bone marrow, umbilical cords, placentas, adipose tissue, dermal skin, skeletal muscle, hair follicles, synovial membranes, and teeth1-5. Currently, attention has been given to synovial-derived MSCs because these cells may help treat joint diseases, such as bone fraction, osteoarthritis, and rheumatoid arthritis (RA), and due to the regenerative potential of MSCs in damaged cartilage or bone, they may also help treat immune modulation or autoimmune diseases6-8. The majority of research of MSCs derived from synovial sources have employed MSCs from the synovial membrane rather than from synovial fluid2,9; thus, the biological understanding of synovial-fluid-derived MSCs is limited.
Synovial fluid is an easily accessible source of MSCs, which can be aspirated during the diagnoses or treatments of patients and during diagnostic confirmations of various arthritis conditions that do not require invasive procedures, such as osteoarthritis or RA. The population of synovial-fluid-derived MSCs in the synovial cavity is significantly increased in arthritic patients compared to non-arthritic patients10-12. Therefore, synovial fluid is an excellent source candidate for MSCs, particularly for autologous stem cell therapy in patients with inflamed or injured joints. Additionally, synovial-fluid-derived MSCs have a chondrogenic capacity, immunomodulation abilities, and a high rate of proliferation. This study established protocols for bone marrow extraction and synovial fluid aspiration from minipigs using non-invasive methods and for the in vitro expansion of isolated cells and analysis of cell phenotypes.

<table border="0" cellpadding="0" cellspacing="0" width="499">
	<tbody>
		<tr height="70">
			<td height="70" style="height:70px;width:136px;">Advanced Dulbecco&#8217;s modified Eagle medium (ADMEM)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">12491-023</td>
			<td style="width:143px;">MSC culture medium</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Dulbecco&#8217;s phosphate-buffered saline (DPBS)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">14190-144</td>
			<td style="width:143px;">Cell washing medium, free of Ca2+/Mg2+,</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:136px;">Fetal bovine serum (FBS)</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">16000-044</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:136px;">Glutamax</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">35050-061</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Penicillin/streptomycin</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">10378-016</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Basic fibroblast growth factor (bFGF)</td>
			<td style="width:143px;">Simga</td>
			<td style="width:79px;">F0291</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Bovine serum albumin (BSA)</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">A6003</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Trypsin-EDTA</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:79px;">25200-072</td>
			<td style="width:143px;">Cell dissociation reagent&#160;</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:136px;">&#946;-mercaptoethanol</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">M7522</td>
			<td style="width:143px;">Component of MSCs medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:136px;">Isotype antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 550616</td>
			<td style="width:143px;">Isotype Control</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:136px;">CD29 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 552369</td>
			<td style="width:143px;">Integrin beta-1 MSCs marker</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:136px;">CD44 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 553133</td>
			<td style="width:143px;">Cell surface glycoprotein MSCs marker</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">CD45 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 340664</td>
			<td style="width:143px;">Hematopoietic stem cells marker</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">CD34 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 555821</td>
			<td style="width:143px;">Hematopoietic stem cells marker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:136px;">CD90 antibody</td>
			<td style="width:143px;">BD Pharmingen</td>
			<td style="width:79px;">BD 555595</td>
			<td style="width:143px;">Thy-1 membrane glycoprotein MSCs marker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:136px;">MHC Class II antibody</td>
			<td style="width:143px;">Santa Cruz</td>
			<td style="width:79px;">SC-32247</td>
			<td style="width:143px;">Antigen presenting cells marker</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Vimentin antibody</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">Sigma-S6389</td>
			<td style="width:143px;">Type III intermediate filament MSCs marker</td>
		</tr>
		<tr height="115">
			<td height="115" style="height:115px;width:136px;">Alkaline phosphatase</td>
			<td style="width:143px;">Promega</td>
			<td style="width:79px;">S3841</td>
			<td style="width:143px;">Mixture of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and nitro blue tetrazolium (NBT)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:136px;">Ficoll-Paque</td>
			<td style="width:143px;">GE Healthcare</td>
			<td style="width:79px;">17-1440-02</td>
			<td style="width:143px;">Density gradient centrifugation</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Cell strainer</td>
			<td style="width:143px;">BD Falcon</td>
			<td style="width:79px;">352340</td>
			<td style="width:143px;">40&#181;m nylon cell strainer</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">15-mL polystyrene conical tube</td>
			<td style="width:143px;">BD Falcon</td>
			<td style="width:79px;">351095</td>
			<td style="width:143px;">Sample collection and cell isolation</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:136px;">35 mm dishes</td>
			<td style="width:143px;">Nunc</td>
			<td style="width:79px;">153066</td>
			<td style="width:143px;">Cell culture dish</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:136px;">Bone marrow extractor</td>
			<td style="width:143px;">GmbH Medizintechnologie, Germany</td>
			<td style="width:79px;">1145-1W010</td>
			<td style="width:143px;">TrokaBone, 3.0x100mm</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Hematocritchamber</td>
			<td style="width:143px;">Marinfeld</td>
			<td style="width:79px;">640130</td>
			<td style="width:143px;">Cell counting chamber</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:136px;">Atropine</td>
			<td style="width:143px;">Jeil Pharmaceutical Co, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Atropine sulfate Hydrate</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:136px;">Acepromazine</td>
			<td style="width:143px;">Samu Median, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Pre-anaesthetic sedative and antiemetic drug</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Medetomidine</td>
			<td style="width:143px;">Pfizer</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anesthetic and analgesic drug</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:136px;">Enrofloxacin</td>
			<td style="width:143px;">Bayer Healthcare, Germany</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anti-biotics</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">Meloxicam</td>
			<td style="width:143px;">Over Veterinary Medicine, Argentina</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Anti-inflammatory and analgesic drug</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Isoflurane</td>
			<td style="width:143px;">Hana pharm, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Inhalational anesthesia</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:136px;">Povidone iodine&#160;</td>
			<td style="width:143px;">Korea Pharma, South Korea</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Sterilization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:136px;">Ethanol</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">E7023</td>
			<td style="width:143px;">Sterilization agent</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:136px;">Heparin</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">H3393</td>
			<td style="width:143px;">Anti-coagulating agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:136px;">Formaldehyde</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:79px;">F8775</td>
			<td style="width:143px;">Cell fixation agent</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:136px;">Ophthalmologic ointment&#160;</td>
			<td style="width:143px;">Pfizer</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Oxytetracycline HCL with polymyxin B sulfate</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:136px;">Circulating water blanket</td>
			<td style="width:143px;">Gaymar Industries</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Warming system during and after anesthesia</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:136px;">Skin stapler</td>
			<td style="width:143px;">Covidien, USA</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Suture skin closure</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:136px;">CO2 Incubator</td>
			<td style="width:143px;">Thermo Forma</td>
			<td style="width:79px;">3111</td>
			<td style="width:143px;">Cell culture Equipment</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:136px;">Flow cytometer</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:79px;"></td>
			<td style="width:143px;">Cell analyzer</td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;width:136px;">Minipig</td>
			<td style="width:143px;">PWG Genetics Korea, Ltd.</td>
			<td style="width:79px;">T-type</td>
			<td style="width:143px;">Miniature pig</td>
		</tr>
	</tbody>
</table>

isolation and cellular phenotyping of mesenchymal stem cells derived from synovial fluid and bone marrow of minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

ミニブタの骨髄および滑液由来MSCの設立 MSCは、正常（ 図2）骨髄およびミニブタの関節滑膜関節から単離され、 インビトロで増殖させました。滑液のシリンジ吸引が簡単であり、初代細胞のインビトロ培養中に十分な接着性細胞を得ることが可能です。滑膜流体由来MSCの形態は、?...

Watch this Scientific Journal Video about Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs at JoVE.com

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

ミニブタの滑液および骨髄由来の間葉系幹細胞の単離および細胞表現型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

isolation-cellular-phenotyping-mesenchymal-stem-cells-derived-from

Research

JoVE Journal

Medicine

10.3K ビュー.  Gyeongsang National University. A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

ビデオ: Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

CD133の患者由来の細胞培養および分離<sup&gt; +</supメラノーマから&gt;推定されるがん幹細胞

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

急性白血病の治療のためのチロシンキナーゼ阻害剤の前臨床評価

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

馬腱でテクネチウム-99m標識骨髄間葉系幹細胞のin vivoイメージングと追跡中

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Isolation of Adipose Derived Regenerative Cells for the Treatment of Erectile Dysfunction Following Radical Prostatectomy

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem cells can be obtained from different tissues and several methods for isolation have been described. The presented method for the isolation of adipose-derived regenerative cells (ADRCs) can be used within many therapeutic areas because the method is a general procedure and, therefore, not limited to erectile dysfunction (ED) therapy. ED is a common and serious side effect to radical prostatectomy (RP) since ED often is not well treated with conventional therapy. Using ADRC&#8217;s as treatment for ED has attracted great interest due to the initial positive results after a single injection of cells into the corpora cavernosum. The method used for the isolation of ADRC&#8217;s is a simple, automated process, that is reproducible and ensures a uniform product. Furthermore, the sterility of the isolated product is ensured because the entire process takes place in a closed system. It is important to minimize the risk of contamination and infection since the stem cells are used for injection in humans. The whole procedure can be done within 2.5-3.5 hours and does not require a classified laboratory which eliminates the need for shipping tissue to an off-site. However, the procedure has some limitations since the minimum amount of drained lipoaspirate for the isolation device to function is 100 g.

Precise disclosure of methods and protocols is crucial for large scale uptake of stem cell therapies. Here, we present a protocol to isolate adipose-derived regenerative cells, used for a single intracavernous injection as treatment of erectile dysfunction (ED) following radical prostatectomy (RP).

根治的前立腺切除術後の勃起不全治療のための脂肪由来再生細胞の単離

Stem cells are used in many research areas within regenerative medicine in part because these treatments can be curative rather than symptomatic. Stem ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

ヒト脂肪由来間葉系幹細胞の単離・増殖のための酵素フリー法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

変形性関節症モデルにおけるマウス脾臓、骨髄、リンパ節および滑膜組織における免疫細胞サブセットのフローサイトメトリー解析

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

ラットにおける骨髄間葉系幹細胞の単離と培養

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Synovial Fluid Analysis to Identify Osteoarthritis

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white blood cell (WBC) count, help define the non-inflammatory nature of SF, which is a hallmark of OA. In patients with OA, WBC in SF samples usually does not exceed 2000 cells per microliter, and the percentage of inflammatory cells, such as neutrophils, is very low or absent. Calcium crystals are frequent in SF collected from OA patients. Although their role in the pathogenesis of OA remains unclear, they have been associated with a mild inflammatory process and a more severe disease progression. Recently, calcium crystals have been described in both the early and late stages of OA, indicating that they may play a vital role in diagnosing different clinical subsets of OA and pharmacological treatment. The overall goal of SF analysis in OA is two-fold: to ascertain the non-inflammatory degree of SF and to highlight the presence of calcium crystals.

Synovial fluid analysis under transmitted, polarized, and compensated light microscopy is used to evaluate the inflammatory or non-inflammatory nature of a sample through simple steps. It is particularly useful in osteoarthritis to detect calcium crystals and identify a more severe subset of osteoarthritis.

変形性関節症を特定するための滑液分析

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

ヒト異種異系フリーのヒト治療法によるヒト脂肪由来幹細胞の単離と培養

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...