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. חומרי הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן 10 חיץ HEPES מ&quot;מ. התאם את ה- pH 7.0 ב RT ולהוסיף NaCl 0.9%. </li><li style=";text-align:right;direction:rtl"> הכן 5% פתרון אלגינט ופתרון 10 מ&quot;מ EDTA על ידי ערבוב מלח HEPES שנאגר ערוכים 1.1. התאם את ה- pH 7.0 ב RT. </li><li style=";text-align:right;direction:rtl"> החיטוי חומרים כימיים (1.1, 1.2) במשך 20 דקות ב 121 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> להכין מאכלי 60 מ&quot;מ מצופה ג&#39;לטין שכבתי עם 1.2-2.0 פיברובלסטים עכבר עובריים × 10 6 (MEF) תאים, אשר נחשבים כתאים מזינים על ידי דגירה בתוך DMEM המכיל 10% FBS ES-מוסמך ו -10 מיקרוגרם / מיליליטר של C mitomycin 90 - 120 דקות. </li><li style=";text-align:right;direction:rtl"> לשמור על גזע pluripotent עכבר מושרה (iPS) תאים בצלחת עם התאים מזינים בנוכחות 5 מיליליטר הגבוה של גלוקוז DMEM המכיל 20% של ES-המוסמך FBS, 50 מיקרומטר של 2-mercaptoethanol, חומצת אמינו לא חיונית, ואנטיביוטיקה ( 100 / מיליליטר יחידות של פניצילין G נתרן, 100 מיקרוגרם / מיליליטר של סולפט סטרפטומיצין, ו -250 / מיליליטר ng של amphoteriCIN B). </li><li style=";text-align:right;direction:rtl"> זרע 4 × 5 תאי iPS 10 על צלחת 60 מ&quot;מ מצופה ג&#39;לטין ודגירתם על 37 מעלות צלזיוס, 5% CO 2. לשנות את מדיום התרבות בכל יום במהלך דגירה. לאחר 3-4 ימים של תרבות, trypsinize תאי דקות 1 עם 500 μl של 0.05% טריפסין המכיל EDTA 0.02% על 37 מעלות צלזיוס, 5% CO 2. אחרי זה, לנתק את התאים על ידי הקשה על צלחת התרבות עשר פעמים. </li><li style=";text-align:right;direction:rtl"> להוסיף 2.5 מיליליטר של DMEM עם 10% מהשעית תא צנטריפוגות ES-מוסמכת FBS ואנטיביוטיקה (להלן תאי iPS עכבר ניתוק לתוך תאים בודדים ידי pipetting עם micropipette 1000 μl שלוש פעמים) בשעה 160 × גרם במשך 3 דקות ולהסיר supernatant ולספור את התאים . בעקבות ספירת תאים, לזרוע תאים שנאספו על צלחת ג&#39;לטין מצופה ו דגירה למשך 30 דקות לבודד את תאי iPS מתאי MEF. לאחר הספירה של התאים (בדרך כלל 1-3 × 10 6 תאים / מנה יכולה להיות שנאספו), reseed 4 × 10 5 תאים iPS עלהצלחת. </li></ol> 2. תא Encapsulation לאלגינט Hydrogel קפסולות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאסוף את התאים על ידי אותו ההליך מתואר ב1.5 ו -1.6. בעקבות צנטריפוגה 160 × גרם במשך 3 דקות, לשטוף את התא גלולה iPS עם מי מלח HEPES שנאגר שלוש פעמים. לאחר מחדש השעיית התאים בתמיסת מלח HEPES שנאגר, לסנן את ההשעיה התא דרך מסננת 40 מיקרומטר תא כדי להסיר אגרגטים גדולים, שעלולים לסתום את החרירים. </li><li style=";text-align:right;direction:rtl"> מערבבים 2 מיליליטר של השעיה תא בתוך תמיסת מלח HEPES שנאגר ו 3 מיליליטר של 5% פתרון ALG-Na. לבסוף 5 מיליליטר של השעיה תא בתוך 3% פתרון ALG-Na מתקבל. צפיפות תאים היא desirably יותר מ 10 6 תאים / מיליליטר. </li><li style=";text-align:right;direction:rtl"> לאחר איסוף ההשעיה התא לתוך מזרק 5 מיליליטר, להתקין זרבובית שיתוף צירי (איור 1 א &#39;, ב&#39;) על המזרק ולהגדיר אותם על משאבת מזרק. זרימת גז 2 לפלוט N (נמוך מ1 ליטר / דקה) באמצעות המחט החיצונית של נחיר קואקס ולגרש את ההשעיה התא דרך הנחיר הפנימי. </li><li style=";text-align:right;direction:rtl"> לאסוף טיפות לתוך 250 מיליליטר של 0.5% פתרון CaCl 2 ולהמתין 10 - 20 דקות לgelation תוך ערבוב ב60-90 סל&quot;ד. </li></ol> 3. טיפול באלגינט קפסולות (אופציונאלי) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה כמוסות ALG-Ca ב0.05% (w / v) פתרון פולי-L ליזין (PLL) במשך 5 דקות ב 37 מעלות צלזיוס, 5% CO 2. </li><li style=";text-align:right;direction:rtl"> לאחר שטיפת הכמוסות עם מי מלח HEPES שנאגר, דגירתם בDMEM המכיל 10% FBS כדי לנטרל את המטען החשמלי על פני השטח שלהם. לנצל אותם ככמוסות מצופים (איור 1 ג). </li><li style=";text-align:right;direction:rtl"> דגירה הכמוסות במלח HEPES שנאגר המכיל 10 mM EDTA במשך 5 דקות. הכמוסות טופל EDTA מנוצלות ככמוסות חלולות (איור 1 ג). </li></ol> 4. תאי תרבות ואיסוף בקפסולות אלגינט <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה התאים במארז ב 75 סל&quot;ד עם רעד על 37 מעלות צלזיוס, 5% CO 2במשך 10 ימים. </li><li style=";text-align:right;direction:rtl"> לאסוף דגירה הכמוסות ב 10 מ&quot;מ EDTA על 37 מעלות צלזיוס, 5% CO 2 למשך 10 דקות. איסוף תאים מכמוסות אלגינט ללא טיפול PLL על ידי צנטריפוגה בז × 1,000 3 דקות. </li><li style=";text-align:right;direction:rtl"> אם מטופלים כמוסות אלגינט עם PLL, לשבור את קרום ALG-PLL ידי pipetting למעלה ולמטה כעשר פעמים עם מחט מחוברת למזרק ו צנטריפוגות זה ב 1000 - 5000 × גרם במשך 5 דקות. קוטר מחט צריך להיות נמוך יותר מגודל כמוסה ו -25 G מחטים (260 מיקרומטר) משמשות בניסוי זה (600 מיקרומטר) </li><li style=";text-align:right;direction:rtl"> לאחר צנטריפוגה, לאסוף תא גלולה לחלוטין מקרומי ALG-PLL שבור אם אתה רוצה לאסוף דגימות ה- mRNA מהתאים. קרומי ALG-PLL נותרו אולי למנוע טיהור mRNA. </li></ol>

<ol>
	<li>Zweigerdt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+scale+production+of+stem+cells+and+their+derivatives.">Large scale production of stem cells and their derivatives.</a> Adv. Biochem. Eng. Biotechnol. 114, 201-235 (2009).</li><li>Chen, A., Chen, X., Choo, A. B. H., Reuveny, S., Oh, S. K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+microcarrier+properties+affecting+the+expansion+of+undifferentiated+human+embryonic+stem+cells.">Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells.</a> Stem cell Res. 7 (2), 97-111 (2011).</li><li>Schroeder, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+lineage+selection+of+mouse+embryonic+stem+cells+in+a+stirred+bench+scale+bioreactor+with+automated+process+control.">Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control.</a> Biotechnol. Bioeng. 92 (7), 920-933 (2005).</li><li>Leung, H. W., Chen, A., Choo, A. B. H., Reuveny, S., Oh, S. K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agitation+can+induce+differentiation+of+human+pluripotent+stem+cells+in+microcarrier+cultures.">Agitation can induce differentiation of human pluripotent stem cells in microcarrier cultures.</a> Tissue Eng. Part C. 17 (2), 165-172 (2011).</li><li>Dang, S. M., Gerecht-Nir, S., Chen, J., Itskovitz-Eldor, J., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled,+scalable+embryonic+stem+cell+differentiation+culture.">Controlled, scalable embryonic stem cell differentiation culture.</a> Stem Cells. 22 (3), 275-282 (2004).</li><li>Weber, L. M., He, J., Haskins, K., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEG-based+hydrogels+as+an+in+vitro+encapsulation+platform+for+testing+controlled+for+testing+controlled+&#223;-cell+microenvironments.">PEG-based hydrogels as an in vitro encapsulation platform for testing controlled for testing controlled &#223;-cell microenvironments.</a> Acta Biomater. 2 (1), 1-8 (2006).</li><li>Horiguchi, I., Chowdhury, M. M., Sakai, Y., Tabata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+morphology,+and+pluripotency+of+mouse+induced+pluripotent+stem+cells+in+three+different+types+of+alginate+beads+for+mass+production.">Proliferation, morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production.</a> Biotechnol. Prog. 30 (4), 896-904 (2014).</li><li>Rahman, N., Purpura, K. A., Wylie, R. G., Zandstra, P. W., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+vascular+endothelial+growth+factor+functionalized+agarose+to+guide+pluripotent+stem+cell+aggregates+toward+blood+progenitor+cells.">The use of vascular endothelial growth factor functionalized agarose to guide pluripotent stem cell aggregates toward blood progenitor cells.</a> Biomaterials. 31 (32), 8262-8270 (2010).</li><li>Siti-Ismail, N., Bishop, A. E., Polak, J. M., Mantalaris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+benefit+of+human+embryonic+stem+cell+encapsulation+for+prolonged+feeder-free+maintenance.">The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance.</a> Biomaterials. 29, 3946-3952 (2008).</li><li>Magyer, J. P., Nemir, M., Ehler, E., Suter, N., Perriard, J., Eppenberger, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Production+of+Embryoid+Bodies+in+Microbeads.">Mass Production of Embryoid Bodies in Microbeads.</a> Ann. N. Y. Acad. Sci. 944, 135-143 (2001).</li><li>Xu, J., Li, S., Tan, J., Luo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controllable+Preparation+of+Monodispersed+Calcium+Alginate+Microbeads+in+a+Novel+Microfluidic+System.">Controllable Preparation of Monodispersed Calcium Alginate Microbeads in a Novel Microfluidic System.</a> Chem. Eng. Technol. 31 (8), 1223-1226 (2008).</li><li>Sakai, M. P., Y, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Bioactive+Hydrogel+Capsules+for+The+3D+Expansion+of+Pluripotent+Stem+Cells+in+Bioreactors.">Development of Bioactive Hydrogel Capsules for The 3D Expansion of Pluripotent Stem Cells in Bioreactors.</a> Biomater. Sci. 2 (176), 176-183 (2014).</li><li>Chowdhury, M. M., Katsuda, T., Montagne, K., Kimura, H., Kojima, N., Akutsu, H., Ochiya, T., Fujii, T., Sakai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+effects+of+secreted+soluble+factor+preserve+better+pluripotent+state+of+embry-+onic+stem+cell+culture+in+a+membrane-based+compartmentalized+micro-bioreactor.">Enhanced effects of secreted soluble factor preserve better pluripotent state of embry- onic stem cell culture in a membrane-based compartmentalized micro-bioreactor.</a> Biomed. Microdevices. 12 (6), 1097-1105 (2010).</li><li>Chowdhury, M. M., Kimura, H., Fujii, T., Sakai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+alternative+fate+other+than+default+neuronal+fate+of+embryonic+stem+cells+in+a+membrane-based+two-chambered+micro-+bioreactor+by+cell-secreted+BMP4.">Induction of alternative fate other than default neuronal fate of embryonic stem cells in a membrane-based two-chambered micro- bioreactor by cell-secreted BMP4.</a> Biomicrofluidics. 6 (1), 14117-14117-13 (2012).</li></ol>

The authors have nothing to disclose.

<html> תרבות Encapsulation ניתן להשוות עם תרבויות השעיה ישירות. תרבות השעיה היא שיטה פשוטה כדי להשיג כמויות גדולות של תאי גזע pluripotent מאשר שיטות אנקפסולציה. עם זאת, השליטה הצבירה של תאים בתרבית השעיה היא עדיין מאתגרת. בשיטת אנקפסולציה, צבירה סלולרית מוגבלת בכמוסות וניתן לשלוט ו?...

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.

בפרוטוקול 2.5, פתרון אלגינט גורש יוצר צורה כדורית מייד לאחר הגירוש (איור 2 א - ח). אם ההשעיה הוא גורש עם קצב זרימה N 2 נמוך יותר מאשר 1 ליטר / דקה, גודל הטיפות הוא אחיד (איור ט 2). עם זאת, אם את זרימת N 2 היא גבוהה יותר מאשר 1 ליטר / דקה, הטיפה מתפרקת <stro...

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.

Encapsulation אלגינט של תאי גזע pluripotent שימוש נחיר Co-הצירית

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

alginate-encapsulation-pluripotent-stem-cells-using-co-axial

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

12.6K Views.  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.

Video: 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.

אלגינט microcapsule כפלטפורמה 3D עבור ריבוי והתמיינות של בתאי גזע עובריים אנושיים (hESC) כדי שושלות שונות

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

Bioengineering

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.

קצב העברת נתונים גבוה תא יחיד ו מרובה תאים מיקרו אנקפסולציה

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.

Encapsulation נייד ב הידרוג 3D עבור הנדסת רקמות

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

Biology

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.

טכניקת Dispensing רובוטיות אוטומטית להכוון משטחים Bioprinting של תאים

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.

אנקפסולציה תא יונקים ב Alginate חרוזים באמצעות כלי פשוט מתערבל

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

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>יצירת כדורי רקמות באמצעות מכשיר דמוי בול מודפס בתלת מימד

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

יצירת כדורי רקמות באמצעות מכשיר דמוי בול מודפס בתלת מימד

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Biochemistry

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.

ליבה/פגז הדפסה פיגומים קפלים להנדסת רקמות של מבנים צינורי

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.

ייצור מיקרופלואידי של מיקרו-מעטפת ליבה הנושאים כדוריות תאי גזע פלוריפוטנטיים אנושיים

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.

תמיכה מרוכבת במטריצה חוץ-תאית מיקרוג'ל להדפסה תלת-ממדית משובצת של מבנים עצביים אנושיים

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 ...

Encapsulation אלגינט של תאי גזע pluripotent שימוש נחיר Co-הצירית

Summary

Explore More Videos

Chapters in this video

אלגינט microcapsule כפלטפורמה 3D עבור ריבוי והתמיינות של בתאי גזע עובריים אנושיים (hESC) כדי שושלות שונות

קצב העברת נתונים גבוה תא יחיד ו מרובה תאים מיקרו אנקפסולציה

Encapsulation נייד ב הידרוג 3D עבור הנדסת רקמות

טכניקת Dispensing רובוטיות אוטומטית להכוון משטחים Bioprinting של תאים

אנקפסולציה תא יונקים ב Alginate חרוזים באמצעות כלי פשוט מתערבל

יצירת כדורי רקמות באמצעות מכשיר דמוי בול מודפס בתלת מימד

יצירת כדורי רקמות באמצעות מכשיר דמוי בול מודפס בתלת מימד

ליבה/פגז הדפסה פיגומים קפלים להנדסת רקמות של מבנים צינורי

ייצור מיקרופלואידי של מיקרו-מעטפת ליבה הנושאים כדוריות תאי גזע פלוריפוטנטיים אנושיים

תמיכה מרוכבת במטריצה חוץ-תאית מיקרוג'ל להדפסה תלת-ממדית משובצת של מבנים עצביים אנושיים