Name: electrically conductive scaffold to modulate and deliver stem cells
Uploaded: 2018-04-13T13:30:00.000+00:00
Duration: 5 min 49 s
Description: Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

Agradecemos o Dr. Kati Andreasson (departamento de Neurologia e ciência neurológica, Universidade de Stanford) para uso da máquina qRT-PCR. O trabalho foi apoiado pela nacional institutos de saúde subsídios K08NS098876 (para P.M.G.) e Postdoctoral Fellowship do reitor de Stanford School of Medicine (ao b.o. n).

Todas as células-tronco e procedimentos animais foram aprovados pelo Comitê de supervisão de pesquisa de células-tronco de Stanford e pelo painel administrativo da Universidade de Stanford em cuidado de Animal de laboratório (SCRO-616 e APLAC-31909).
1. gravura de vidro de ITO
<ol><li>Preparar o óxido da lata do indium (ITO)-lâmina de vidro coberta pela colocação de um agente de mascaramento no lado do condutor do vidro. Nota: Mais comerciais fitas transparentes podem ser usadas como um agente de mascaramento.<ol>
<li>Remover a máscara em excesso, usando uma lâmina, deixar apenas a forma desejada do design final ITO coberto no vidro.</li>
	</ol>
</li>
	<li>Combine 5% (v/v) de ácido nítrico e 45% (v/v) de HCl num copo de vidro dentro de uma coifa para preparar a solução de gravura.<ol>
<li>Usar um prato quente e um termômetro para garantir que a temperatura da solução de condicionamento é mantida a 70 ° C.</li>
	</ol>
</li>
	<li>Uma vez que a solução de gravura chega a 70 ° C, coloque a lâmina de vidro de mascarados-ITO na solução por 2 minutos remover a camada de ITO em excesso. Nota: Mascaramento de fita protege a camada de ITO de exposição a solução ácida.</li>
	<li>Retire o slide da solução e enxágue com deionizada (DI) H2O.</li>
	<li>Remova cuidadosamente a máscara e medida de resistência usando um multímetro para garantir que os restante ITO está intacto. Leituras da área desejada gravada devem ser de aproximadamente 0 Ω.</li>
</ol>2. preparação da solução de pirrol
<ol><li>Uma garrafa de vidro de 1000 mL, dissolver 70 g de dodecylbenzenesulfonate de sódio (NaDBS) em 600 mL de DI H2O.</li>
	<li>Uma vez que o NaDBS é dissolvido, adicione 14 mL de pirrol 0,2 M e 386 mL de DI H2O para a solução.</li>
	<li>Cobrir a solução com papel alumínio e armazene a 4 ° C.</li>
</ol>3. galvanização de polipirrol em vidro de ITO
<ol><li>Aquecer a solução de pirrol à temperatura até NaDBS é totalmente evapora. Nota: Isto leva cerca de 15 a 20 minutos.</li>
	<li>Despeje uma proveta 25 mL da solução de pirrol.</li>
	<li>Conectar o vidro de ITO gravado a um multímetro e mergulhe o slide a solução de pirrol.<ol>
<li>Certifique-se de que o lado com a resistência de Ω 0 é virada para o eléctrodo de referência de malha de platina.</li>
	</ol>
</li>
	<li>Aplica uma corrente elétrica para iniciar a galvanização do PPA na superfície de ITO, usando um multímetro (corrente controlada em 3 mA/cm2 para 4 horas).</li>
	<li>Remova o multímetro e lave galvanizada-PPA em DI H2O surfactante residual de remover o vidro de ITO.</li>
	<li>Suavemente retire a chapa galvanizada-PPA do vidro de ITO, usando uma lâmina de barbear.<ol>
<li>Armazene a placa PPA à temperatura ambiente.</li>
	</ol>
</li>
</ol>4. preparação do Polydimethylsiloxane (PDMS)
<ol><li>Usando uma espátula e um prato de pesagem, misturar 18 g de agente de base com 2 g de agente de cura e despeje a mistura em moldes de vidro dois 10 x 8 cm.</li>
	<li>Remova as bolhas de moldes utilizando uma câmara de vácuo para 15-20 minutos.</li>
	<li>Coloque os moldes em um forno de 70 ° C por 3 h.</li>
	<li>Uma vez arrefecido, use uma espátula plana para remover o PDMS de moldes.</li>
</ol>5. fabricação da câmara de estimulação elétrica em Vitro
<ol><li>Autoclave a placas de metal (WxL, 2,5 cm x 12,5 cm) e fluxo de válvulas a 120 ° C por 1 hora.</li>
	<li>Usando um slide da câmara como um modelo, corte dois pedaços de PDMS. Nota: Um pedaço de PDMS serve como o perímetro da câmara de célula com recortes de dois quadrados contíguos de dentro da câmara de célula. O outro pedaço PDMS é o contorno do perímetro da câmara.<ol>
<li>Cortar o interior câmara de célula para que ele seja quadrado em forma de e coincide com a forma da câmara de célula. Certifique-se que a forma geral de ambas as partes de PDMS é retangular e correspondências do slide a câmara.</li>
	</ol>
</li>
	<li>Os materiais da camada seguinte de baixo para cima: (1) placa, de Metal (2) PDMS (sem recortes), prato (3) PPA (perpendicular ao PDMS), (4) PDMS (com recortes) e câmara de celular (5).</li>
	<li>Fixe o aparelho juntos uma vez que ele é totalmente alinhado.</li>
	<li>Corte dois pedaços de 60 cm de um fio de metal e use o colar de prata para anexar um fio para cada extremidade da placa PPA que se projeta do lado de fora da câmara. Certifique-se de que os fios são tempo suficiente para estender do aparelho para o exterior de uma incubadora.</li>
	<li>Uma vez que o colar de prata está curado, reforçar e selar a conexão do fio com epóxi.<ol>
<li>A resistência usando um multímetro para certificar-se de que o campo aplicado é o mesmo em cada câmara de registro.</li>
		<li>Para implantação, retire os fios; desapertar as câmaras de célula e PDMS e, em seguida, eles separam o andaime condutora. A dimensão dos andaimes implantado é de aproximadamente 1 x 3 x 0,25 mm.</li>
	</ol>
</li>
</ol>6. chapeamento humano neurais progenitoras (hNPCs) no PPA
<ol><li>Esterilize as câmaras montadas sob luz UV durante 2 horas.<ol>
<li>Depois de 1 hora, gire o montado câmaras de 90°.</li>
	</ol>
</li>
	<li>Após a esterilização, reveste a superfície do PPA na parte inferior da câmara com 800 µ l de um substrato de revestimento e permitir que o substrato solidificar na placa PPA na incubadora a 37 ° C em 5% de CO2 por 1h.</li>
	<li>Depois de 1 hora, retire o sobrenadante das câmaras e lave delicadamente com 1 mL de DPBS + Ca + Mg por bem. Nota: Evite lavar a superfície do PPA com força, porque isso poderá provocar o desprendimento do substrato do revestimento.</li>
	<li>Usando a mídia de células frescas, mecanicamente dissocia pilhas cultivadas para uso com as câmaras de uma placa de cultura de tecido com suave pipetagem. Nota: As células devem ser confluente de 90-100% em cima de dissociação.</li>
	<li>Coletar hNPC pelotas usando centrifugação a 8000 x g, durante 5 minutos.</li>
	<li>Usando um hemocytometer, contar e ressuspender as células em um volume de 100.000 células/cm2.</li>
	<li>Placa de 100.000 células em cada câmara bem (100.000 células/cm2 em 1 mL de mídia).</li>
	<li>Câmaras de retornar a incubadora a 37 ° C em 5% CO2.
</li>
</ol>7. eletroestimulação de hNPCs
<ol><li>Um dia após a semeadura as células sobre as câmaras, use um gerador de forma de onda para aplicar estimulação elétrica para as células.</li>
	<li>Antes da estimulação, remover a velha mídia de cada câmara bem e reabasteça com 800 µ l de mídia fresca.</li>
	<li>Depois que a mídia é alterada, colocar câmaras de volta para a incubadora, estender os cabos fora da incubadora e anexá-los a um gerador de forma de onda</li>
	<li>Aplica o estímulo para as células com um sinal de onda quadrada em mV ±400 com 100 Hz por 1h.</li>
	<li>Após a estimulação, retire os fios e incubar as câmaras para outro dia em uma incubadora que defina a 37 ° C com 5% de CO2.</li>
	<li>Realizar análise biológica subsequente, incluindo a viabilidade celular e reação em cadeia da polimerase em tempo real quantitativa (qRT-PCR) seguindo o protocolo do fabricante.</li>
</ol>8. na Vivo PPy implantação
<ol><li>Executar modelos de stroke de oclusão de artéria cerebral média distal (dMCA) na deficiência de células T ratos nus macho adulto (NIH-RNU 230 ± 30 g) como descrito anteriormente2. Realize a oclusão de post-dMCA de uma semana de implantação.</li>
	<li>Um dia antes da cirurgia, dê ampicilina (1 mg/mL) para os ratos em sua água de gaiola.</li>
	<li>Anestesiar o rato em uma câmara de indução usando 0,5 - 3% mg/kg de isoflurano administrado por inalação.</li>
	<li>Confirme anesthetization do rato pela falta de reflexo resposta pitada de dedo do pé.<ol>
<li>Enquanto o animal está sob anestesia, continue a monitorar sua membrana cor, padrão de respiração e temperatura retal a cada 15 minutos.</li>
	</ol>
</li>
	<li>Uma vez confirmada anesthetization, aplica lágrimas artificiais aos olhos do rato para evitar ressecamento.</li>
	<li>Garantir a manutenção técnica asséptica durante este procedimento, usando luvas estéreis e um pano cirúrgico estéril13. Manter estéril instrumentos cirúrgicos dentro de um campo estéril.</li>
	<li>Enquanto o rato está sob anestesia, perfure uma craniectomia acima o córtex esquerdo. Abra a dura-máter.<ol>
<li>Remova a dura-máter do cérebro usando uma agulha fina micro cirúrgica.</li>
	</ol>
</li>
	<li>Implante os andaimes condutoras do sistema in vitro para o córtex de rato. Nota: Para nossos experimentos, implantamos andaimes em vitro dia 3 após hNPC chapeamento.<ol>
<li>Coloque o implante principalmente no córtex penumbral medial à lesão de acidente vascular cerebral.</li>
	</ol>
</li>
	<li>Após o andaime é colocado, coloque surgicel sobre o implante para impedir a movimentação com o fechamento da pele.</li>
	<li>Sutura ferida fechada e injectar por via subcutânea os ratos com buprenorfina SR a uma dosagem de 1 mg/kg para controlar a dor associada com cirurgia estereotáxica e oclusão de dMCA.</li>
	<li>Coloque o rato em uma gaiola de recuperação como ele recobra a consciência.<ol>
<li>Nunca abandone o animal enquanto está inconsciente.</li>
		<li>Não coloque o animal com os outros até totalmente ganhou consciência.</li>
	</ol>
</li>
	<li>Monitorar os animais diariamente para sinais de dor, incluindo perda de peso corporal, ingestão de comida/água diminuiu, casaco de aliciamento/despenteado reduzido, aumento/diminuição da atividade espontânea, postura anormal, desidratação/pele acampar, olhos encovados, escondido, auto-mutilação, respiração rápida, respiração de boca aberta, tremendo, tremendo, tremor e vocalização.<ol>
<li>Monitorar os animais diariamente, até parece que eles estão comendo, bebendo, aliciamento e ganhando peso; e então semanal após o ganho de peso.</li>
	</ol>
</li>
	<li>Eutanásia precoce através da inalação de CO2 e uma confirmação secundária de eutanásia (assegurar o animal não reviverá devido por inalação de oxigênio normal abastecimento post CO2 ) ocorrerá a qualquer animal que aparenta não estar comendo, bebendo e ganho de peso após o procedimento cirúrgico inicial; aparece na dor; ou parece incapaz de concluir os testes comportamentais devido a um maior que o esperado déficit do córtex motor.</li>
</ol>

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Os autores têm sem conflitos de interesse, para declarar-se com este trabalho.

Crescentes evidências tem demonstrado a promessa de células-tronco como uma terapia de romance, acidente vascular cerebral. Esta promessa resultou em um grande esforço para avançar terapêutica de células-tronco para o lado da cama, pelo menos 40 ensaios clínicos em curso ou já concluídos. Patologia de curso oferece um único distúrbio neurológico que se presta a terapia de células-tronco, porque após o insulto agudo, não há nenhum processo neurodegenerativas, impedindo a recuperação. O mecanismo exato de...

Derrame é a segunda principal causa de morte no mundo e a quinta causa principal de morte nos Estados Unidos. Apesar destas altas taxas de morte, tratamentos para recuperação de acidente vascular cerebral atualmente permanecem um desafio com sem opções viáveis de médicos actualmente disponíveis1. Existem atualmente cerca de 300 ensaios clínicos lidando com traços isquêmicos, dos quais somente 40 utilizam células-tronco. Estudos anteriores demonstraram que as terapias de células-tronco têm um efeito benéfico sobre o curso reparo2,3. Parácrina fatores como fator neurotrófico derivado do cérebro (BDNF) e thrombospondin-1 (THBS-1) lançado a partir de células progenitoras neurais humanas transplantadas (hNPCs) têm mostrado recuperação funcional melhorada através de mecanismos associados com um aumento na sinapse formação, angiogênese, ramificações dendríticas e novas projeções axonal, bem como modulando o sistema imunológico4,5,6. No entanto, os métodos de entrega ideal das células-tronco permanecem indescritíveis.
Entrega de células-tronco bem sucedida para o cérebro continua a ser um desafio. Atualmente, hidrogel injetável e sistemas poliméricos andaimes foram introduzidos para fornecer células-tronco. Esses métodos de entrega protegem transplante de células-tronco, bem como oferecem proteção contra o ambiente áspero de acidente vascular cerebral incluindo resposta inflamatória do hospedeiro e as condições de hipóxia7,8,9 , 10. no entanto, os materiais mais comumente usados são inertes, que limita o uso de modulação contínua (ou seja, estimulação elétrica) das células11. Estimulação elétrica é uma sugestão que influencia a diferenciação e densidade de canais de íon axônio consequência de células-tronco12. Em comparação com os polímeros inertes, polímeros condutores podem transportar um atual permitindo a estimulação elétrica e manipulação de células-tronco2. No entanto, o mecanismo preciso pelo qual estimulação elétrica modula a liberação de fator neurotrophic (i.e., BDNF e THBS-1) é ainda não totalmente explorado.
Este protocolo, descreveremos os passos para construir um sistema de cultura de células consistindo de um andaime de polímero condutor, polipirrol (PPA), que permite a estimulação elétrica em vitro . Por causa da maneira em que o sistema de cultura de células é fabricado, a posterior implantação de células-tronco-semeado cadafalso para o córtex peri-infarto é possível. Para este sistema, temos condição eletricamente as células-tronco no cadafalso por um curto período de tempo antes da implantação. Após a estimulação elétrica, o andaime de polímero condutor carregando as células é implantado com sucesso intracraniano, usando um método minimamente invasivo.

<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

Terapia de células-tronco tem emergido como um excitante curso terapêutica, mas o método de entrega ideal permanece obscuro. Enquanto a técnica de microinjeção tem sido usada há décadas para fornecer células-tronco em modelos stroke, esta técnica é limitada pela falta de habilidade para manipular as células-tronco antes da injeção. Este documento detalha um método do uso de um andaime de polímero eletricamente condutivo para a entrega de células-tronco. Estimulação elétrica de células-tronco usando um andaime de polímero condutor altera os genes da célula tronco envolvidos na sobrevivência celular, resposta inflamatória e remodelamento sináptico. Após pré-condicionamento elétrico, as células-tronco no andaime são transplantadas intracraniano em um modelo de rato de oclusão de artéria cerebral média distal. Este protocolo descreve uma técnica poderosa para manipular as células-tronco através de um andaime de polímero condutor e cria uma nova ferramenta para desenvolver a terapia baseada em células-tronco.

O diagrama mostrado na Figura 1 representa o fluxo de trabalho geral da estimulação elétrica de hNPCs e potenciais aplicações a jusante. Uma limitação atual na terapia de células-tronco é que as células-tronco são expostas a um ambiente áspero do pós-transplante incluindo inflamação e condições isquêmicas. Estas condições difíceis prováveis limitam sua eficácia terapêutica14,15...

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

Este protocolo descreve a fabricação de um sistema de cultura de células para permitir a propagação de células-tronco em um andaime de polímero condutor para estimulação elétrica em vitro e subsequentes na vivo implantação da célula-tronco-semeado andaime usando um técnica minimamente invasiva.

Eletricamente condutivo Andaime modular e entregar as células-tronco

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

12.7K visualizações.  Stanford University School of Medicine. Este protocolo descreve a fabricação de um sistema de cultura de células para permitir a propagação de células-tronco em um andaime de polímero condutor para estimulação elétrica em vitro e subsequentes na vivo implantação da célula-tronco-semeado andaime usando um técnica minimamente invasiva.

Vídeo: 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.

Uso de Células-tronco humanas perivasculares para Regeneração Óssea

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

Bioengenharia

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.

Engenharia de Tecidos: Construção de um multicelular 3D andaime para a entrega de camadas celulares 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 ...

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.

Otimizando Penhora de mesenquimais humanas Células-Tronco em Poly (ε-caprolactona) electrospun Yarns

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.

Electrospun nanofibras Andaimes com gradações de Organização Fiber

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.

3D Agregação Stem Magnetic celular e biorreator Maturação para regeneração da cartilagem

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

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.

Estimulação simultânea de elétrica e mecânica para aumentar o potencial de Cardiomyogenic das células

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.

Construção e utilização de uma câmara de estimulação elétrica para reforçar a diferenciação osteogênica em células estromais/tronco mesenquimais em Vitro

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.

Preparação de Microambientes de Matriz Extracelular Incapaz para avaliar especificação de fenótipo celular schwann

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.

Modelagem de elementos finitos de um microambiente elétrico celular

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

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

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

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

Otimizando a entrega de vesículas extracelulares usando um andaime bioimpresso em 3D com bainha central para tratamento de feridas crônicas

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