Dr. Kati Andreasson (Nöroloji ve nörolojik bilimler, Stanford Üniversitesi) qRT-PCR makinesi kullanımı için teşekkür ederiz. İş Ulusal kurumları K08NS098876 sağlık hibe (için P.M.G.) ve Stanford Tıp Fakültesi Dekan'ın doktora sonrası bursu (için Bo) tarafından desteklenmiştir.

Tüm kök hücre ve hayvan yordamlar Stanford'un kök hücre araştırma Gözetim Komitesi tarafından ve laboratuvar hayvan bakım (SCRO-616 ve APLAC-31909) Stanford Üniversitesi'nin Yönetim Panel tarafından kabul edildi.
1. gravür Ito cam
<ol><li>İndiyum kalay oksit (ITO) hazırlamak-cam iletken yan tarafa bir maskeleme ajanı yerleştirerek kaplı cam kaymak. Not: Çoğu ticari şeffaf bantlar bir maskeleme ajanı olarak kullanılabilir.<ol>
<li>Bir bıçak kullanarak aşırı maskesi kaldırmak için yalnızca istediğiniz şekli son ITO tasarım bırakarak cam kaplı.</li>
	</ol>
</li>
	<li>Nitrik asit (v/v) %5 ve % 45'i (v/v) HCl bir duman başlık içinde bir cam ölçek büyütme çözüm hazırlamak için birleştirir.<ol>
<li>Çözüm aşındırma sıcaklık 70 ° C'de korunur emin olmak için elektrikli Ocak ve bir termometre kullanın</li>
	</ol>
</li>
	<li>70 ° C Dağlama çözüm ulaştıktan sonra maskeli-ITO cam slayt 2 dakika fazla ITO kat kaldırmak çözüm içine yerleştirin. Not: Bant maskeleme asit solüsyonu maruz ITO katman korur.</li>
	<li>Slayt çözümden kaldırmak ve deiyonize (DI) H2ile O. durulayın</li>
	<li>Kalan ITO sağlam olduğundan emin olmak için bir multimetre kullanarak maske ve ölçü direnç dikkatli bir şekilde çıkarın. Sonuçlar istenilen kazınmış alanının yaklaşık 0 Ω olmalıdır.</li>
</ol>2. Pyrrole çözüm hazırlanması
<ol><li>1000 mL cam şişe 70 g sodyum dodecylbenzenesulfonate (NaDBS) 600 mL DI H2O. geçiyoruz.</li>
	<li>Bir kez NaDBS çözülür, 14 mL 0,2 M pyrrole ve DI H2O 386 mL ekleyin.</li>
	<li>Alüminyum folyo ile çözüm kapak ve 4 ° C'de depolayın</li>
</ol>3. galvanik Polypyrrole ITO camına,
<ol><li>NaDBS tamamen redissolved kadar oda sıcaklığında pyrrole çözümünü sıcak. Not: Bu yaklaşık 15-20 dakika sürer.</li>
	<li>25 mL pyrrole çözeltisi bir kabı dökün.</li>
	<li>Kazınmış ITO cam için bir multimetre bağlanmak ve slayt pyrrole çözüm içine daldırın.<ol>
<li>0 Ω direnci ile yüzün Platin kafes referans elektrot doğru dönük olduğundan emin olun.</li>
	</ol>
</li>
	<li>Bir elektrik akımı bir multimetre (3 mA/cm2 ' de 4 saat boyunca kontrollü akım) kullanarak ITO yüzeyinde PPy galvanik başlatmak için geçerlidir.</li>
	<li>ITO cam multimetre ve yıkama alaşımlı-PPy kalan yüzey aktif kaldırmak için DI H2O kaldırın.</li>
	<li>Yavaşça bir jilet kullanarak ITO cam alaşımlı PPy tabaktan ayırın.<ol>
<li>PPy plaka oda sıcaklığında saklayın.</li>
	</ol>
</li>
</ol>4. Polydimethylsiloxane (PDMS) hazırlanması
<ol><li>Bir spatula ve tartmak çanak kullanarak, temel Aracısı 18 g kür Ajan 2 g ile mix ve iki 10 cm x 8 cm cam kalıpları içine karışımı dökün.</li>
	<li>Kabarcıklar vakum odası 15-20 dakika kullanarak kalıplar kaldırın.</li>
	<li>Kalıpları 3 h 70 ° C fırında yerleştirin.</li>
	<li>Soğutmalı sonra düz bir spatula PDMS kalıpları kaldırmak için kullanabilirsiniz.</li>
</ol>5. Vitro elektriksel stimülasyon odası imalatı
<ol><li>Otoklav metal levhalar (WxL, 2.5 cm x 12,5 cm) ve akışı vanalar 120 ° C'de 1 saattir.</li>
	<li>Odası slayt şablon olarak kullanıp, iki adet PDMS kesti. Not: Tek parça PDMS hücre odası çevre kesik iki bitişik kareler içinde hücre odası ile hizmet vermektedir. Diğer PDMS odası 's çevre bir taslağını parçasıdır.<ol>
<li>Kesmek iç hücre odası böylece kare şeklinde ve hücre odası şeklinde eşleşir. PDMS iki adet genel şekli dikdörtgen ve odası slayt uygun emin olun.</li>
	</ol>
</li>
	<li>Malzeme--dan dip aşağıdakine üst olarak katman: (1) Metal plaka, (2) PDMS (olmadan kesik), (3) PPy plaka (PDMS için dikey), PDMS (4) (ile kesik) ve (5) hücre odası.</li>
	<li>Bir kez tam olarak hizalanır aparatı birlikte kelepçe.</li>
	<li>Bir metal tel iki 60 cm parçalar koparıp ve gümüş hamur odası dışarıdan protrudes PPy plaka her sonu için bir kablo bağlamak için kullanın. Teller cihaz bir kuluçka dışa doğru genişletmek yeterince uzun olduğundan emin olun.</li>
	<li>Bir kez gümüş Yapıştır tedavi güçlendirmek ve epoksi ile tel bağlantı mühür.<ol>
<li>Direnç uygulanan alanını her odasında aynı olduğundan emin olmak için bir multimetre kullanarak kaydedin.</li>
		<li>İmplantasyon için kabloları çıkarın; hücre odaları unclamp ve iletken iskele PDMS ve sonra onları ayrı. İmplante iskele yaklaşık 1 x 3 x 0,25 mm boyuttur.</li>
	</ol>
</li>
</ol>6. insan nöral progenitör hücre (hNPCs) PPy kaplama
<ol><li>UV ışık altında birleştirilmiş chambers sterilize etmek için 2 saat.<ol>
<li>1 saat sonra monte döndürün 90 ° Odaları.</li>
	</ol>
</li>
	<li>Sterilizasyon sonra PPy odası alt kaplama substrat 800 µL ile yüzey kat ve 1 h için % 5 CO2 37 ° C'de bir kuluçka PPy tabakta kuvvetlendirmek belgili tanımlık substrate sağlar.</li>
	<li>1 saat sonra süpernatant odaları kaldır ve yavaşça DPBS + Ca + Mg 1 mL de başı ile durulayın. Not: Bu kaplama substrat dekolmanı neden olur gibi zorla PPy yüzey yıkama kaçının.</li>
	<li>Taze hücre medyayı kullanarak, mekanik olarak kültürlü hücreler nazik pipetting ile doku kültürü plaka chambers ile kullanmak için ayırmak. Not: Hücreleri % 90-100 birleşmesi ayrılma üzerine olmalıdır.</li>
	<li>Santrifüjü kullanarak de 8000 x g 5 dakika hNPC granül toplamak.</li>
	<li>Bir hemasitometre kullanarak, say ve hücreleri 100.000 hücreler/cm2ses seviyesinde resuspend.</li>
	<li>Her odaya iyi (100.000 hücreler/cm2 ' medya 1 mL) 100.000 hücreleri plaka.</li>
	<li>Kuluçka makinesi %5 37 ° C'de Chambers dönmek CO2.
</li>
</ol>7. elektrik hNPCs uyarılması
<ol><li>Bir gün chambers, hücrelerdeyse tohum sonra bir dalga formu jeneratör elektrik stimülasyonu hücrelere uygulamak için kullanın.</li>
	<li>Stimülasyon önce eski medya de her odasından kaldırın ve taze medya 800 µL ile doldurmak.</li>
	<li>Medya değiştirildikten sonra odaları da kuluçka yerleştirin, kuluçka makinesi dışında teller genişletmek ve onları bir dalga formu jeneratör takmak</li>
	<li>Bir kare dalga sinyali için 1 h 100 Hz ile ±400 mV, hücrelerle stimülasyon uygulanır.</li>
	<li>Stimülasyon sonra kabloları çıkarın ve odaları ile % 5 CO237 ° C'de ayarla bir kuluçka başka bir gün için kuluçkaya.</li>
	<li>Hücre canlılığı ve nicel gerçek zamanlı Polimeraz zincir tepkimesi (PCR qRT) üreticinin protokol sonrası dahil olmak üzere sonraki biyolojik çözümlemesi gerçekleştirin.</li>
</ol>8. içinde Vivo PPy implantasyon
<ol><li>Distal orta serebral arter (dMCA) tıkanıklığı felç modelleri T hücre eksikliği üzerinde gerçekleştirmek yetişkin erkek çıplak rats (NIH-RNU 230 ± 30 g) olarak açıklanan daha önce2. İmplantasyon bir hafta sonrası dMCA tıkanıklığı gerçekleştirin.</li>
	<li>Bir gün önce ameliyat, onların kafes suda sıçanlar için Ampisilin (1 mg/mL) ver.</li>
	<li>Fareyi kullanarak bir indüksiyon odası içinde anestezi 0.5 - % 3 mg/kg isoflurane inhalasyon yolu ile yönetilen.</li>
	<li>Anesthetization farenin burun çimdik refleks yanıt alınamaması tarafından onaylayın.<ol>
<li>Hayvan anestezi altında olmakla birlikte, onun membran renk, nefes desen ve rektal sıcaklık 15 dakikada izlemeye devam edin.</li>
	</ol>
</li>
	<li>Anesthetization onayladıktan sonra suni gözyaşı kuruluğu önlemek için fare gözünü uygulanır.</li>
	<li>Aseptik teknik steril eldiven ve steril cerrahi örtüsü13kullanarak bu yordamı sırasında korunur emin olun. Steril cerrahi aletlerin steril alan içinde tutun.</li>
	<li>Fareyi anestezi altında olmakla birlikte, kraynektomi yukarıda sol korteks matkap. Beyin zarı açın.<ol>
<li>Dura mater beyin cerrahi mikro-ince iğne kullanarak kaldırın.</li>
	</ol>
</li>
	<li>Sıçan korteks vitro sisteminden iletken iskele implant. Not: bizim deneyler için iskele vitro gün 3 hNPC kaplama sonra implante.<ol>
<li>İmplant öncelikle halkalı korteks üzerinde medial kontur lezyon için yerleştirin.</li>
	</ol>
</li>
	<li>İskele yerleştirildikten sonra surgicel hareket cilt kapatma ile önlemek için implant üzerine getirin.</li>
	<li>Yara kapa ve subkutan buprenorfin SR, stereotaksik cerrahi ve dMCA tıkanıklığı ile ilgili ağrı yönetmek için 1 mg/kg doz ile farelere enjekte dikiş.</li>
	<li>Bilinci yerine gibi fare kurtarma kafese koyun.<ol>
<li>Asla baygın durumdayken hayvan çalıştırın.</li>
		<li>O tamamen bilinç kazanmıştır kadar başkaları ile hayvan koymayın.</li>
	</ol>
</li>
	<li>Hayvanlar günlük vücut kilo kaybı, düşük gıda/su alımı, azaltılmış bakım/dağınık ceket, spontan aktivite azaldı/arttı, anormal postür, dehidratasyon/cilt çadır, batık gözler, gizleme, öz sünneti de dahil olmak üzere ağrı belirtileri için izlemek, Hızlı solunum, açık ağızlı nefes, seğirmesi, titreyen, tremor ve seslendirme.<ol>
<li>Onlar yeme, içme, damat ve kilo olduğunu görünene kadar hayvanlar her gün izlemek; ve sonra haftalık sonra kilo.</li>
	</ol>
</li>
	<li>CO2 inhalasyon ve euthanization (hayvan değil normal oksijen kaynağı yazı CO2 inhalasyon nedeniyle canlandırmak sağlamak için) bir ikincil onay ile erken euthanization içme yemek değil, görünür herhangi bir hayvan gerçekleşir ve ilk cerrahi işlem sonrası kilo; acı içinde görünür; veya davranış testleri büyük bir motor korteks açığı beklenenden daha nedeniyle tamamlayamadı görünür.</li>
</ol>

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Yazarlar yok bu iş ile bildirmek için çıkar çatışması var.

Kanıt büyüyen kök hücre vaadi bir roman kontur terapi olarak göstermiştir. Bu söz kök hücre therapeutics yatağımın en az 40 tamamlanmış veya devam eden klinik çalışmalar ile ilerlemek için önemli bir çalışma sonuçlandı. Kontur patoloji çünkü akut hakaret sonra kurtarma engelleyen hiçbir nörodejeneratif işlem ödünç vermek kendisi için kök hücre tedavisi benzersiz bir nörolojik bozukluk sunmaktadır. Kök hücre gelişmiş inme kurtarma tam mekanizmasının belirsizdir. Anjiogenez, syn...

İnme dünyada ölüm ikinci önde gelen nedenidir ve ölüm beşinci önde gelen nedenidir Amerika Birleşik Devletleri olduğunu. Bu yüksek ölüm oranlarını rağmen tedaviler inme kurtarma için şu anda bir sorun yok uygun tıbbi seçenekler mevcut1kalır. Orada şu anda yaklaşık 300 klinik çalışmalarda hangi kök hücreler sadece 40 kullanmak iskemik inme ile ilgili. Önceki çalışmalarda kök hücre tedavileri kontur onarım2,3üzerinde yararlı bir etkisi olduğunu ortaya koymuştur. Parakrin faktörler beyin kaynaklı Nörotrofik faktör (BDNF) ve Transplante insan nöral progenitör hücre (hNPCs) yayımlanan trombospondin-1 (THBS-1) gibi bir artış ile ilgili mekanizmalar yoluyla geliştirilmiş fonksiyonel iyileşme göstermiştir oluşumu, anjiogenez, dendritik dallanma ve yeni aksonal projeksiyonlar sinaps gibi bağışıklık sistemi4,5,6modülasyonlu. Ancak, kök hücrelerin en uygun teslimat yöntemlerinin zor kalır.
Başarılı kök hücre teslim beyin için bir meydan okuma olarak kalır. Şu anda, enjekte edilebilir hidrojel ve polimerik İskele sistemleri kök hücre sunmak için getirilmiştir. Bu teslim yöntemi kök hücre nakli sırasında korumak hem de ana bilgisayarın inflamatuar yanıt ve hipoksik koşullar7,8,9 gibi sert inme sonrası çevre koruma , 10. ancak, en yaygın olarak kullanılan malzemeler hangi hücreleri11sürekli Modülasyon (Yani, elektriksel stimülasyon) kısıtlar etkisiz,. Elektriksel stimülasyon farklılaşma, iyon kanal yoğunluğu ve kök hücre12neurite akıbet etkiler bir ipucudur. İnert polimerler ile karşılaştırıldığında, elektriksel stimülasyon ve kök hücre2manipülasyon için geçerli bir izin iletken polimerler taşıyabilir. Ancak, hangi tarafından Nörotrofik faktör sürümü (Yani, BDNF ve THBS-1) elektriksel stimülasyon modüle kesin mekanizması hala tam keşfedilmeden.
Bu protokol için bir iletken polimer iskele, vitro için elektriksel stimülasyon sağlar polypyrrole (PPy), oluşan bir hücre kültür sistemi oluşturmak için adımları açıklar. Hangi hücre kültür sistemi fabrikasyon olduğunu şekilde nedeniyle, peri-enfarktüsü korteks üzerine kök hücre numaralı seribaşı iskele sonraki implantasyonu mümkündür. Bu sistem için biz elektrikle kök hücre iskele üzerinde kısa bir süre önce implantasyon için yaygõn. Elektriksel stimülasyon hücreleri taşıyan iletken polimer iskele intracranially minimal invaziv bir yöntem kullanarak başarılı bir şekilde yerleştirilir.

<table><tbody><tr><td>FGF-Basic</td><td>Invitrogen</td><td>CTP0261</td><td>20 ng/mL for working media</td></tr><tr><td>Matrigel</td><td>Corning</td><td>cb40234a</td><td>1:200 dilution</td></tr><tr><td>LIF Protein, Recom. Hum. (10 &amp;micro;g/mL)</td><td>EMD Millipore</td><td>LIF1010</td><td>10 ng/mL for working media</td></tr><tr><td>Sylgard 184 silicone 3.9 kg</td><td>Fisherbrand</td><td>NC0162601</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Fisherbrand</td><td>SA56-4</td><td></td></tr><tr><td>Nitric Acid Concentrate (Certified) ACS, Fisher Chemical</td><td>Fisherbrand</td><td>SA95</td><td></td></tr><tr><td>ITO Glass</td><td>Delta Technologies</td><td>CG-40IN-0115</td><td></td></tr><tr><td>Sodium dodecylbenzenesulfonate</td><td>Sigma</td><td>289957-1KG</td><td></td></tr><tr><td>Pyrrole</td><td>Sigma</td><td>131709-500ML</td><td>Protect pyrrole solution from light and room temperature</td></tr><tr><td>8 well glass slide chambers</td><td>Thermo Sci Nuc&amp;nbsp;</td><td>125658</td><td>Detach the cell chamber and keep it under sterilized conditions</td></tr><tr><td>Flat-Surface Bracket, 3&quot;x1&quot;</td><td>McMaster-Carr&amp;nbsp;</td><td>1030A4</td><td></td></tr><tr><td>TWO PART SILVER PAINT 14G</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>1264214</td><td>Mix two parts (1:1) in plastic plate</td></tr><tr><td>DPBS, 1x, with Ca and Mg, No Phenol Red</td><td>Genesse&amp;nbsp;</td><td>25-508C</td><td></td></tr><tr><td>AB2 ArunA Neural Cell Culture Media Kit</td><td>Aruna Biomedical&amp;nbsp;</td><td>ABNS7013.2</td><td></td></tr><tr><td>hNP1 Human Neural Progenitor Expansion Kit</td><td>Aruna Biomedical&amp;nbsp;</td><td>&amp;nbsp;hNP7013.1</td><td></td></tr><tr><td>Noncontact Flow-Adjustment Valve, Nickel-Plated Brass, for 3/32&quot; to 5/8&quot; Tube OD</td><td>McMaster-Carr&amp;nbsp;</td><td>5330K22</td><td></td></tr><tr><td>Multimeter</td><td>Keysight&amp;nbsp;</td><td>E3641A</td><td></td></tr><tr><td>Wavefoam generator</td><td>Keysight</td><td>33210A-10MHz</td><td></td></tr><tr><td>Pt meshes</td><td>Sigma-Aldrich&amp;nbsp;</td><td>298107-425MG</td><td>Reference electrode with dimensions, 1x1 cm</td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>L3224</td><td></td></tr><tr><td>BDNF</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs02718934_s1</td><td></td></tr><tr><td>THBS1</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs00962908_m1</td><td></td></tr><tr><td>GAPDH</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>Hs02758991_g1</td><td></td></tr><tr><td>RNeasy Mini Kit (250)</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>QIAshredder (250)</td><td>Qiagen</td><td>79656</td><td></td></tr><tr><td>RNase-Free DNase Set (50)</td><td>QIAGEN</td><td>79254</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit, 100 x 20 &amp;micro;L rxns</td><td>BIORAD</td><td>1708891</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>4369510</td><td></td></tr><tr><td>7-8 Week Old, male, RNU Rats</td><td>NCI-Frederick</td><td></td><td></td></tr><tr><td>4-0 Ethicon Silk Suture</td><td>eSutures.com</td><td>683G</td><td></td></tr><tr><td>Isoflurane</td><td>Henry Schein</td><td>29405</td><td></td></tr><tr><td>V-1 Tabletop Laboratory Animal Anesthesia System</td><td>VetEquip</td><td>901806</td><td></td></tr><tr><td>Surgicel Original Absorbable Hemostat</td><td>Ethicon</td><td>1952</td><td></td></tr><tr><td>Lab Standard Stereotaxic Instrument, Rat</td><td>Stoelting</td><td>51600</td><td></td></tr><tr><td>Kimberly-Clark Professional&amp;nbsp;Safeskin Purple Nitrile Sterile Exam Gloves</td><td>Fisherbrand</td><td>19-063-130</td><td></td></tr><tr><td>Sterile Drape</td><td>Medline</td><td>DYNJSD1092</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Shandon Stainless-Steel Scalpel Blade Handle, Holds No. 20-25 Blades</td><td>Fisherbrand</td><td>53-34</td><td></td></tr><tr><td>Walter Stern Scalpel Blade Series 300</td><td>Fisherbrand</td><td>17-654-456</td><td></td></tr><tr><td>QuantStudio 6 Flex Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>4484642</td><td></td></tr><tr><td>Frazier Micro Dissecting Hook</td><td>Harvard Apparatus</td><td>52-2706</td><td></td></tr></tbody></table>

electrically conductive scaffold to modulate and deliver stem cells

Kök hücre tedavisi heyecan verici bir kontur olarak terapötik çıkmıştır ama en uygun Teslimat yöntemi belirsizdir. Mikroenjeksiyon tekniği kök hücre kontur modellerinde sunmak için yıllardır kullanılan iken, bu teknik enjeksiyon önce kök hücre işleme yeteneği eksikliği ile sınırlıdır. Bu kağıt bir elektriksel olarak iletken polimer iskele kök hücre teslim için kullanma yöntemi ayrıntıları. Bir iletken polimer iskele kullanarak kök hücrelerin elektriksel stimülasyon kök hücrenin genler hücre hayatta kalma, inflamatuar yanıt ve sinaptik remodeling yer değiştirir. Elektrik Önkoşullanma sonra iskele üzerinde kök hücre intracranially distal orta serebral arter tıkanıklığı sıçan modelinde nakledilen. Bu iletişim kuralı bir iletken polimer iskele üzerinden kök hücre işlemek için güçlü bir teknik anlatılmaktadır ve daha fazla kök hücre tabanlı terapisi geliştirmek için yeni bir araç oluşturur.

Şekil 1 ' de gösterilen şematik genel iş akışını hNPCs ve potansiyel aşağı akım uygulamaları elektriksel stimülasyon ve temsil eder. Bir geçerli kök hücre tedavisi, kök hücre iltihabı ve iskemik koşulları gibi sert bir sonrası nakli ortamına maruz kısıtlamadır. Bu zor koşullar büyük olasılıkla onların tedavi edici etkinliği14,15sınırlayın. Bu ortamdan hNPCs korumak...

Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Bu iletişim kuralı bir iletken polimer iskele vitro elektriksel stimülasyon ve kök hücre numaralı seribaşı iskele kullanarak sonraki vivo içinde implantasyon için üzerinde kök hücrelerin tohum izin vermek için bir hücre kültür sistemi imalatı açıklar bir minimal invaziv tekniği.

Modüle ve kök hücre sunmak için elektriksel olarak iletken iskele

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

electrically-conductive-scaffold-to-modulate-and-deliver-stem-cells

Research

JoVE Journal

Bioengineering

12.8K Görüntüleme.  Stanford University School of Medicine. Bu iletişim kuralı bir iletken polimer iskele vitro elektriksel stimülasyon ve kök hücre numaralı seribaşı iskele kullanarak sonraki vivo içinde implantasyon için üzerinde kök hücrelerin tohum izin vermek için bir hücre kültür sistemi imalatı açıklar bir minimal invaziv tekniği.

Video: Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Kemik Rejenerasyon için İnsan perivasküler Kök Hücre kullanın

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Biyomühendislik

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Doku Mühendisliği: Katmanlı Hücre Sheets teslimi için bir Multisellüler 3D İskele İnşaatı

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Poli (ε-kaprolakton) electrospun İplik İnsan Mezenkimal Kök Hücre Ek Optimize

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Fiber Teşkilatı geçişleri ile electrospun Nanolif iskeleleri

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Kıkırdak Yenileme için 3D Manyetik Kök Hücre Toplama ve Biyoreaktör Olgunlaşma

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

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.

3D dinamik kültür sistemi ile bir çok katmanlı mezenkimal kök hücre levha inşaatı

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 ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Hücrelerin Cardiomyogenic potansiyeli geliştirmek için aynı anda elektrik ve mekanik stimülasyon

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

İnşaat ve in Vitro mezenkimal kök/Stromal hücrelerdeki Osteojenik farklılaşma güçlendirmeye yönelik bir elektriksel stimülasyon odası kullanımı

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Schwann Hücre Fenotip Spesifikasyonunu Değerlendirmek Için Hücre Dışı Matriks Mikroortamlarının Hazırlanması

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.