This research was supported by the S-Innovation project of the Japan Science and technology Agency (JST), the Graduate Program for Leaders in Life Innovation (GPLLI) of the University of Tokyo, and the Research Fellowship for Young Scientists of Japan Society for the Promotion of Science. We thank nac Image Technology Inc. for taking movies using a hi-speed camera.

1. 준비 재료 <ol><li> 10 mM의 HEPES 버퍼를 준비합니다. 실온에서 7.0으로 pH를 조정하고 0.9 %의 염화나트륨을 추가합니다. </li><li> 1.1 준비 HEPES 완충 식염수를 혼합하여 5 % 알긴산 용액 10 mM의 EDTA 솔루션을 준비합니다. 실온에서 7.0으로 pH를 조절합니다. </li><li> 121 ℃에서 20 분 동안 시약 (1.1, 1.2)를 압력솥. </li><li> DMEM 10 % ES-자격 FBS와 90 마이 토마 이신 C의 10 ㎍ / ㎖의를 포함하는 내 배양 피더 세포로 처리됩니다 2.0 × 10 6 마우스 배아 섬유 아세포 (MEF) 세포, - 1.2 계층화 젤라틴 코팅 60mm 요리를 준비합니다 - 120 분. </li><li> (ES-정규화 FBS 20 % 2- 머 캅토 에탄올, 비 필수 아미노산 및 항생제 50 μM을 함유하는 5 ㎖의 DMEM 고 글루코오스의 존재 하에서 피더 세포와 접시에 세포 (IPS) 마우스 유도 된 다 능성 줄기 유지 페니실린 G 나트륨, 황산 스트렙토 마이신 100 μg의 / ㎖의 100 단위 / ㎖ 및 250 ng를 amphoteri / ㎖CIN의 B). </li><li> 시드 4 × 10 5 만능 줄기 젤라틴 코팅 60mm 접시에 세포 및 CO 2를 37 ° C에서 그들을 품어 5 %. 배양시 배양 배지 매일을 변경합니다. 3 후 - 문화의 4 일, 37 ° C에서 0.05 % 트립신은 0.02 %의 EDTA를 포함하는 500 μL와 1 분 5 % CO 2 세포를 Trypsinize. 그 후, 배양 접시에 10 번을 두드려서 세포를 분리. </li><li> 3 분 동안 160 × g에서 원심 분리기 세포 현탁액 (세 번 1,000 μL 마이크로 피펫과 피펫 팅하여 단일 세포로 분리 마우스 IPS 세포 다음) FBS와 항생제 ES-자격의 10 % DMEM의 2.5 ML을 추가하고 상층 액을 제거하고 세포를 계산 . 셀 카운팅 후, 젤라틴 코팅 접시에 세포를 수집하고 시드 MEF 세포로부터 만능 줄기 세포를 분리하기 위해 30 분 동안 배양한다. 세포를 카운팅 한 후 (일반적으로 1 - × 106 세포 / 접시가 수집 될 수 있음), 4 × 105 IPS 세포를 시드요리. </li></ol> 알긴산 하이드로 겔 캡슐에 2 셀 캡슐화 <ol><li> 1.5 및 1.6에 기재된 방법과 동일한 방법에 의해 세포를 수집. 3 분 동안 160 × g에서 원심 분리 후, 세 번 HEPES 완충 식염수와 유도 만능 줄기 세포 펠렛을 씻는다. HEPES 완충 식염수에 세포를 다시 현탁 한 후, 별도로 노즐을 막히게 할 수있는 큰 응집체를 제거하기 위해 40 μm의 셀 스트레이너를 통해 세포 현탁액을 필터. </li><li> HEPES 완충 식염수 내에서 세포 현탁액 2 ㎖ 및 5 % Δ1g의-나트륨 용액 3 mL를 섞는다. 마지막으로 3 % Δ1g의-나트륨 용액 내 세포 현탁액 5 ml를 얻을 수있다. 셀 밀도는 바람직하게 10 개 이상 6 세포 / ml이다. </li><li> 5 ML의 주사기로 세포 현탁액을 수집 한 후, 주사기에 동축 노즐 (그림 1A, B)를 설치하고 주사기 펌프를 설정합니다. 발광 N 2 가스 유동 (저급 1L / 분 이하) 동축 노즐의 외 바늘 통해내부 노즐을 통해 세포 현탁액을 추방. </li><li> 0.5 % CaCl2를 250 ml의 용액에 방울을 모아서 10 기다려 - 60-90 rpm에서 교반하면서 겔을 20 분. </li></ol> 알긴산 캡슐 3. 치료 (선택 사항) <ol><li> 0.05 %에서 Δ1g의 - 칼슘 캡슐 부화 폴리 -L- 라이신 (PLL) 37 ° C에서 5 분 동안 5 % 용액 (/ V w) CO 2. </li><li> HEPES 완충 식염수와 캡슐을 세척 한 후, DMEM는 그 표면에 전하를 중화하기 위해 10 % FBS를 함유하는 배양에서 그들을. 코팅 캡슐 (그림 1C)로 활용. </li><li> 5 분 동안 10 mM의 EDTA를 함유하는 HEPES 완충 식염수에서 캡슐 부화. EDTA 처리 된 캡슐 빈 캡슐 (그림 1C)로 사용된다. </li></ol> 알긴산 캡슐 4. 문화 및 수집 세포 <ol><li> 37 ℃ 및 5 % CO 2에서 진탕 75 rpm에서 캡슐화 된 세포를 부화십일합니다. </li><li> 수집하고 10 분 동안 CO 2, 37 ℃에서 10 mM의 EDTA에 캡슐을 배양하고, 5 %. 3 분 1,000 × g에서 원심 분리하여 PLL을 처리하지 않고 알긴산 캡슐에서 세포를 수집합니다. </li><li> 5 분 동안 5,000 ×의 G - 알지네이트 캡슐이 PLL로 처리하는 경우, 최대 피펫 아래로 열 배 1,000에서 주사기와 원심 분리기를에 부착 된 바늘에 의해 ALG 파일-PLL 막을 휴식. 니들 직경은 캡슐의 크기 및 25 G (260 μm의) 바늘이 실험에서 사용되는보다 낮아야한다 (600 μm의) </li><li> 당신이 세포에서 mRNA의 샘플을 수집 할 경우 원심 분리 후, 깨진 Δ1g의-PLL 막으로부터 엄격하게 세포 펠렛을 수집합니다. 남은 Δ1g의-PLL 막은 가능한 mRNA의 정제를 방지한다. </li></ol>

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The authors have nothing to disclose.

<html> 캡슐화 배양 직접 현탁 배양 물과 비교 될 수있다. 현탁 배양은 캡슐화 방법보다 다 능성 줄기 세포를 대량으로 얻을 수있는 간단한 방법이다. 그러나, 현탁 배양 세포의 집계를 제어하는​​ 것은 여전히​​ 어렵습니다. 밀봉 방법에서, 세포 응집은 캡슐에 제한되며, 따라서 잘 제어 할 수있다. 이전 간행물은 큰 세포 덩어리가 자유 현탁 배양 7에 등장하는 반면 캡슐화 된 세포, 균일 한 크기의 집계를 형성 것으로 나타났다. 또한, 이전 보고서는 강한 교반 PS 세포의 직접 서스펜션 문화에 세포 죽음을 일으키는 것을 증명; 따라서 제한된 교반 속도를 필요로. 반대로, 심지어 캡슐화 서스펜션 전단 응력 조건에서 세포를 보호 할 수있다. 부유 세포를 성장하는 경우에 밀봉 방법 따라서 운전 조건에 유연성의 수준을 허용한다. &quot;ove_content는&gt; 캡슐화는 양산 공정의 변형뿐만 아니라 줄기 세포 연구 모두에 유용한 도구이다. 줄기 세포의 캡슐화가 세포 외 기질 및 성장 인자를 포함하는 줄기 세포의 틈새를 연구에 유용하다. 일부 연구자는 것을 증명 VEGF 공역 하이드로 겔, 아가 로스로 마우스 ES 세포의 캡슐화는 혈액 선조 세포로 8 세포의 분화를 촉진. 이 문서에서는 쉽게 동축 노즐 및 N 2 흐름을 사용하여 크기 조절 Δ1g의 - 칼슘 캡슐에 마우스 IPS 세포를 캡슐화하는 방법을 보여줍니다. 이러한 연동 펌프 (9) 및 전기 전압 (10)과 같은 다양한 도구와 일부 봉지 공정이 있지만, 유동 N 2를 사용하여 캡슐화는 다른 방법보다 더 간단하고 안전한 방법이다. 또한,이 방법은 (약 600 ㎛의보다 큰) 연동 펌프와 방법보다 더 작은 캡슐을 형성 할 수있다(약보다 큰 3mm). 대형 캡슐은 완전히 겔화 시간이 더 걸릴 CaCl2를 긴 시간 배양 가능성이 세포의 특성에 영향을 미친다. 또한, 큰 캡슐은 또한 아마도 글루코스 및 산소와 같은 영양소 폐기물이나 물질 전달의 문제가있다. 따라서, N 2 흐름 (600 -1,000 μm의)에 의해 형성된 캡슐의 크기는 내부에 세포 배양에 적합한 크기이다. 이 방법은 작은 캡슐을 제조하고 정확하게 크기를 제어하는​​데 적합하지 않다. 왜냐하면 물방울의 파괴,이 방법은 500 ㎛의보다 작은 캡슐을 제조하기에 적합하지 않다. 또한, 캡슐의 크기를 결정할 수있는 유일한 시각적 관찰은, 따라서이 방법에 의해 캡슐의 크기를 제어하는​​ 것이 곤란하다. 이 경우, 마이크로 유체 소자의 사용은 아마도 이러한 문제를 해결할 수있다 (11). Δ1g의 - 나 겔은 CaCl2를 오디오 솔루션 회의 직후 때문에, 때때로 막힘 마이크로 디바이스에서 발생이온. 알긴산이 때문에 고점도의 화학적 개질에 적합하지 않지만, 이는 PEG처럼 밀봉에 사용하는 것도 곤란하고, 겔 캡슐과 하이브리드를 형성 할 수있다. 이전 공보는 RGD 펩티드 공역 PEG-Δ1g의 하이브리드 겔 캡슐 마우스 IPS 세포의 세포 성장 캡슐화 (12)를 향상하는 것이 나타났다. 화학적 수정하지 않고, 단지 겔 캡슐에 캡슐화 세포는 분비 된 요소를 유지하여 줄기 세포 틈새를 수정하는 것으로 예상된다. 실제로, 이전의 연구에서 유지 microbioreactor 분비 된 성장 인자, 줄기 세포의 운명을 조절 13,14 효과적이라고 증명. 캡슐에서 세포의 수집은 여전히​​ 다 능성 줄기 세포의 대량 생산에 밀봉 방법의 응용에 대한 미해결 문제이다. 이전 보고서는 PLL 코팅 k로 필요한 것으로 나타났다7 내부 EEP 마우스 유도 만능 줄기 세포. Δ1g의 - 칼슘 겔 캡슐은 물론 이러한 PLL (프로토콜 3.1-3.2)로서 폴리 양이온으로 코팅하는 경우, 그 중 효소 또는 킬레이트 시약으로 캡슐을 분해하기 어렵다. 이 경우, 바늘 피펫 같은 기계적 방법 (프로토콜 4.3) 요구된다. 원심 아마도 세포 생존에 영향을 미칠 수 있지만, 모은 세포 인해 높은 G 하중으로, 5,000 × g에서 원심 분리에보다 더 세분화 막으로부터 분리 될 수있다. 따라서, 부드럽게 세포 파편을 제거뿐만 아니라 캡슐 파괴의 기술은 캡슐화 기술을 개발하는 것이 중요하다. 좀 더 발달이 세포의 대량 생산이 방법을 적용 할 필요가 있지만, 양쪽 밀봉 양산 유망한 기술이다 ​​줄기 세포 연구.

Induced pluripotent stem cells (iPS cells) are currently the source of intense research due to their role in regenerative medicine. However, huge amounts of cells are required for tissue regeneration. For instance approximately one billion pancreatic cells required for a type 1 diabetic patient1. However, conventional dish culture is only able to obtain 1 &#215; 105 cells/cm2, thus requiring 1 m2 of culture area to obtain enough stem cell-derived pancreatic cells to treat a type 1 diabetic patient. The development of a system for the mass-culture of pluripotent stem cells, such as microcarrier2 and suspension culture is therefore required for regenerative medicine. Suspension culture represents a promising method of mass culture but controlling the aggregation of cells is challenging in direct suspension cultures of human iPS cells3. Indeed, suspended cells are exposed to shear stress, which causes cell damage3 or differentiation4.
Research into hydrogel-based encapsulation has been conducted to solve problems associated with suspension culture. In hydrogel capsules, cells are protected from the flow of the medium. Previous reports have documented the use of various types of hydrogel, including agarose5, PEG6, and alginate (Alg), for cellular encapsulation. Alg-Ca hydrogel is one of the most useful hydrogels for cell encapsulation because Alg&#8722;Ca hydrogel is formed immediately after dropping alginate solution into a CaCl2 solution and is also readily digested by enzymes or chelating reagents. 
Here, we have established a stable alginate encapsulation process for iPS cells using a co-axial nozzle. By using N2 gas flow for forming droplets, it is possible to encapsulate cells into uniform capsules without the need for other reagents such as oil. In this method, the flow rate of N2 and concentration of both CaCl2 and alginate are the major operating conditions affecting the size, shape, and uniformity of capsules. This report demonstrates the optimization of these operating conditions through the use of a hi-speed camera and a microscope.

<table><tbody><tr><td>Mouse embryo fibroblast</td><td>Cell Biolabs</td><td>SNL 76/7</td><td></td></tr><tr><td>Mouse induced pluripotent stem cell</td><td>RIKEN Bio resorce centre</td><td>iPS-MEF-Ng-20D-17</td><td></td></tr><tr><td>DMEM high-glucose</td><td>GIBCO</td><td>11995</td><td></td></tr><tr><td>ES qualified&amp;nbsp; FBS</td><td>GIBCO</td><td>16141079</td><td></td></tr><tr><td>Antibacterial Antibiotics</td><td>GIBCO</td><td>15240</td><td></td></tr><tr><td>Nonessential Amino Acid</td><td>GIBCO</td><td>11140</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>GIBCO</td><td>21985-023</td><td></td></tr><tr><td>ESGRO Leukemia Inhibitory Factor</td><td>Merck Millipore</td><td>ESG1107</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>GIBCO</td><td>25300</td><td></td></tr><tr><td>26 G/16 G needle</td><td>Hoshiseido</td><td></td><td></td></tr><tr><td>10 ml Syringe</td><td>TERUMO</td><td>SS-10ESZ</td><td></td></tr><tr><td>Sodium&amp;nbsp; Chloride</td><td>Wako</td><td>191-01665</td><td></td></tr><tr><td>HEPES</td><td>SIGMA</td><td>H4034</td><td></td></tr><tr><td>Sodium Alginate</td><td>Wako</td><td>194-09955</td><td></td></tr><tr><td>Calcium Chloride</td><td>Wako</td><td>039-00475</td><td></td></tr><tr><td>Poly-L-lysine (MW = 15,000 - 30,000)</td><td>SIGMA</td><td>P7890</td><td></td></tr><tr><td>EDTA</td><td>DOJINDO</td><td>345-01865</td><td></td></tr><tr><td>Sylinge pump</td><td>AS ONE</td><td></td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DM IRB</td><td></td></tr><tr><td>Hispeed camera</td><td>nac image technology</td><td>Memrecam HX-3</td><td></td></tr></tbody></table>

alginate encapsulation of pluripotent stem cells using a co-axial nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

프로토콜 2.5에서 추방 알지네이트 솔루션은 추방 후 즉시 구형 (- H 그림 2A)를 형성한다. 현탁액을 1L / 분 이하 N 2 유량으로 배출되는 경우, 액 적의 크기가 균일 한 (도 2I)이다. N 2 흐름은 1L / 분보다 큰 경우, 액체 방울 (도 2G, 흰색 화살표로)을 분해 및 액 적의 크기가 불균일 (도 2J)를하게된다. 이 주어지면,이 방법을 사용하여 500 ㎛의보다 작은 액 적을 제조하는 것은 곤란하다. Δ1g의-나트륨 (3 % 이하)의 농도가 충분 (도 3E-H, K, I) 인 경우, 겔화 과정 (프로토콜 2.4)에서, Δ1g의 - 나이 CaCl2를 용액에 구형 Δ1g의 - 칼슘 캡슐을 형성 방울입니다. Δ1g의-나트륨 (3 % 이하)의 농도는 낮은 그러나, 비 구형 및 비 캡슐의 매끄러운 형상 (도 3A-D, I) 일으키는코팅 공정에서 문제가있다. -Δ1g의 나트륨 농도가 충분하지 않다하더라도, 구형 캡슐 염화칼슘 2 농도 (도 3J)를 증가시킴으로써 형성 될 수있다. CaCl2를 너무 높은 농도는 세포 성장과 생존 (데이터는 도시되지 않음)이 감소하기 때문에, 50 mM의 CaCl2를 캡슐화에 적합하다. 염화칼슘이 솔루션으로 떨어지는 후, 선택적으로는 겔에 대한 몇 분 동안 기다릴 수 있지만, CaCl2를 너무 오래 배양 때로는 세포 성장에 영향을주기 때문에 우리는 염화칼슘이 솔루션에서 캡슐을 따기 좋습니다. 때때로 당신은 농도가 충분한 경우에도 비 구형 모양의 캡슐을 구하십시오. 이 세포 펠렛을 세척하는 Δ1g의 - 나 용액에 세포를 재현 탁 전에 충분하지 아마 때문이다. 혈청 등의 잔류 시약 가능성 겔화 방지한다. 캡슐화 된 세포가 캡슐하지만 휴대 누출 성장할 수있다 (그림에서 화살표가 붙은도 4a) Δ1g의-PLL 코팅 (그림 4A)없이 캡슐에 발생할 수 있습니다. 세포 덩어리를 함께 각 중공 캡슐 (도 4c)의 하나의 구형 응집체를 형성하는 반면, 마우스 IPS 세포의 경우, 세포는, 각 Δ1g의 - 칼슘 캡슐 (도 4b)에서 디스크 형 응집체를 형성한다. 초기 세포 밀도는 캡슐하지만 낮은 세포 밀도 (105 세포 / ㎖ 겔)에서 응집체의 크기에 영향을 (데이터는 미도시)까지 확장 할 수있는 오류가 발생하지 않는다. 따라서, 106 개 세포 / ml 겔 캡슐화하는 것이 바람직하다. 캡슐로부터 세포를 수집하는, Δ1g의 - 칼슘 하이드로 겔은 ALG 파일-PLL 코팅은 효소 또는 화학 물질을 용해하는 것은 곤란하지만, 알긴산 리아제 또는 킬레이트 시약에 용해 될 수있다. ALG-PLL 따라서 물리적으로 예를 들면, 피펫으로 나눌 수 있어야합니다. 깨진 막은 원심 분리에 의해 세포로부터 해리 될 수있다. 캡슐화 시스템 및 캡슐도 1의 이미지. (A)에 Δ1g의 클린 벤치 - 칼슘 밀봉 용 밀봉 시스템의 이미지. (B) 밀봉 용 동축 노즐의 개략도 이미지. (C)이 보고서에서 얻은 캡슐의 세 가지 유형. <a href="https://www.jove.com/files/ftp_upload/52835/52835fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/52835/52835fig2.jpg" /> N 2 동축 노즐로부터 3 % Δ1g의-나트륨 용액의 액적 형성의 모양 (I, J) -도 2 캡슐의 형태에 N 2 흐름의 효과 연속 이미지 (H).1 L / min으로 흐름 (- D, I), 2 L / 분 (E - H, J). 연속 이미지를 고속 카메라로 촬영했다. 히드로 겔 캡슐 CaCl2를 0.5 %에서 형성된다. <a href="https://www.jove.com/files/ftp_upload/52835/52835fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/52835/52835fig3.jpg" /> Δ1g의 - 나 그림 3. 효과와 CaCl2를 집중 캡슐의 형태에 연속 이미지 (- H). 외관 - Δ1g의 - 나의 (I L)을 CaCl2를 용액에 겔 방울입니다. - (E H)의 50 mM CaCl2를 용액에서 겔화되어 - (D)와 3 %의 연속 이미지에서, Δ1g의 - 나, 2 %의 농도를 갖는 상이한 방울. 연속 이미지는 안녕에 의해 캡처- 속도 카메라. 외관 이미지 Δ1g의 - 나, 2 % (L, J) 3 % ​​(K, L), 및 CaCl2를, 50 mM의 (L, K) 및 500 mM의 상이한 농도로 형성 Δ1g의 - 칼슘 캡슐의 형태 학적 변화를 보여 (J, L). <a href="https://www.jove.com/files/ftp_upload/52835/52835fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/52835/52835fig4.jpg" /> () 벌거 벗은, (:. 캡슐의 세 가지 유형의 - (각각 4 1) 4, 6, 8, 10 일간 배양 만능 줄기 세포 집계 그림 4. 캡슐에 형태론 캡슐화의 마우스 만능 줄기 세포 집합체 현미경 사진 이미지 B) 코팅, 및 (c) 빈 캡슐. <a href="https://www.jove.com/files/ftp_upload/52835/52835fig4large.jpg" target="_blank">를 클릭하십시오</a>여기에이 그림의 더 큰 버전을 볼 수 있습니다.

Watch this Scientific Journal Video about Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle at JoVE.com

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

동축 노즐을 사용하여 다 능성 줄기 세포의 알긴산 캡슐화

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

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Developmental Biology

12.6K 조회수.  The University of Tokyo. We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

비디오: Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

다른 Lineages로 인간 배아 줄기 세포 (hESC)의 전파 및 차별 화를위한 3D 플랫폼으로서 알긴산의 Microcapsule

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

연구

생체공학

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

높은 처리량 마이크로 캡슐 단일 셀 및 다중 셀

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

조직 공학을위한 3D Hydrogels에서 셀룰러 봉지

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

생물학

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

세포의 표면지도 및 Bioprinting에 대한 자동화 된 로봇 분배 기법

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

간단한 교반 용기를 사용하여 알긴산 염 비즈에서 포유류 세포 캡슐화

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

관 구조의 조직 공학을 위한 코어/쉘 인쇄 비계

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 &#181;m diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with &gt;95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had &lt;50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

인간 다능성 줄기 세포 구상체를 운반하는 코어-쉘 마이크로캡슐의 미세유체 제조

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM).
To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs.
First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

인간 신경 구조물의 임베디드 3D 프린팅을 위한 마이크로겔-세포외 기질 복합체 지원

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

포르신 오시테 캡슐화를 위한 프로틸분해 된 알기네이트 하이드로겔 및 소수성 미생물 반응기

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

후성유전학적 변형과 생체역학적 단서를 결합하여 포유류 다능성 세포를 생성하는 두 단계 전략

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...