This work was funded by NIH (U54HD41857 and PL1EB008542, a P30 Biomaterials Core within the Oncofertility Consortium Roadmap grant).

1. Follicle Isolation
Experiments on animals were performed in accordance with the guidelines and regulations set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the established Institutional Animal Use and Care protocol at Northwestern University.
For optimal results, all dissections are carried out in L15 media for pH control at ambient levels of CO2, on 37&deg;C heated stages for temperature control, and on a clean bench to minimize bacterial contamination. The dissection media (DM) is prepared with L15 media supplemented with 50 IU/mL penicillin and 50 &mu;L/mL streptomycin and 1% FBS. The maintenance media (MM) is prepared with &alpha;MEM media supplemented with 50 IU/mL penicillin and 50 &mu;L/mL streptomycin and 1% FBS.
<ol>
 <li>Transfer freshly dissected ovaries from 16 day old mouse into a new 35 mm Petri dish with 1-2 mL MM containing 0.1% Collagenase and 0.1% DNase. Incubate for 15 minutes inside an incubator at 37&deg;C and 5% CO2.</li>
 <li>After incubation, perform several washing steps in DM to remove collagenase, and then transfer to a 35 mm Petri dish with fresh DM. Isolate follicles from the ovary by gently flicking (or cutting) follicles away from the whole ovary by using two 28 5/8 gauge needles attached to disposable syringes. Remove as much stroma as possible without damaging the integrity of follicle. Dissect out 20-40 secondary follicles per ovary (130-150mm). These follicles usually have 2-3 layers of somatic cells. </li>
 <li>Add 1 mL MM to the central well of a 60 mm IVF (in vitro fertilization) Petri dish, and 3 mL MM to the outer ring. Transfer intact follicles with a pipette to the outer ring of the IVF dish to briefly rinse, and then selectively transfer them into the central well (See 1.4 ). Store this IVF dish in the incubator.</li>
 <li>After all the follicles are collected, the selection step is performed under a dissecting microscope with a 5-8x magnification. Healthy follicles have the following morphological characteristics:
 <ul>
 <li>2-3 somatic cell layers</li>
 <li>130-150 mm</li>
 <li>No separation between oocyte and somatic cells</li>
 <li>Intact and round oocytes 
 Transfer the healthy follicles into the center of IVF dishes for encapsulation.</li>
 </ul>
 </li>
</ol>

 
2. Follicle Encapsulation, Method 1 - "The Drop Method"
<ol>
 <li>Add 1 mL of 50 IU/mL thrombin in TBS with 40 mM CaCl2 into the middle well of the IVF dish. Thaw and keep fibrinogen stock (50 mg/mL in TBS) on ice. Bring the fibrinogen to room temperature right before use.</li>
 <li>Prepare the fibrinogen/alginate solution by mixing 1x PBS, 0.5% alginate in PBS solution and 50 mg/mL fibrinogen solution at a 2:1:1 ratio in a 1.7 mL sterile microcentrifuge tube. Avoid introducing bubbles into the solution. Gently vortex and spin-down. The solution appears slightly cloudy and should be prepared immediately before use.</li>
 <li>Place two droplets of fibrinogen/alginate solution in the outer ring of an IVF dish: a 90 &mu;L droplet for encapsulation and a 10 &mu;L droplet for washing. To decrease evaporation, turn off the heating stage on the dissecting microscope. Transfer 10-15 follicles into the 10 &mu;L droplet with a minimal amount of culture media (&lt; 5 &mu;L) using a 200 &mu;m micropipette tip, and quickly mix. Transfer all of the follicles into the 90 &mu;L droplet with a minimal amount of solution (&lt; 5 &mu;L) from the first drop, and mix.</li>
 <li>Simultaneously aspirate one follicle and 5 &mu;L of fibrinogen/alginate solution using a 10 &mu;L pipette tip, and release into the thrombin/Ca2+ solution in the IVF dish. Repeat this step until all the follicles are encapsulated.</li>
 <li>Cross-link the beads for 5-7 minutes in the thrombin/Ca2+ solution. The beads will become darker as fibrin gels. Transfer the beads into a Petri dish containing MM, starting with the darker beads first. Incubate the dish for 15-30 minutes inthe incubator to rinse off the remaining thrombin.</li>
 <li>Transfer the beads into a 96 well culture plate containing 100 &mu;L of follicle growth media, and image immediately. The growth media (GM) is prepared with &alpha;MEM media supplemented with 10 mIU/mL of recombinant FSH , 3 mg/mL of BSA, 1 mg/mL of bovine fetuin, 5 &mu;g/mL of insulin, 5 &mu;g/mL of transferrin, and 5 ng/mL of selenium.</li>
</ol>
3. Follicle Encapsulation, Method 2 - "The Parafilm Method"
<ol>
 <li>Prepare the fibrinogen/alginate solution by mixing 0.5% alginate solution and 50 mg/mL fibrinogen solution at a 1:1 ratio in a 1.7 mL sterile microcentrifuge tube.</li>
 <li>Pipette 7.5 &mu;L drops of the fibrinogen/alginate mixture onto a parafilm coated glass slide with 3 mm spacers. Transfer 1 follicle into each drop with a minimal amount of media.</li>
 <li>Add 7.5 &mu;L of thrombin/Ca2+ solution to each drop. Mixing is unnecessary because the gel forms almost instantaneously.</li>
 <li>Cover the gels with the second parafilm coated glass slide, put the slides upside down in a 100 mm Petri dish, and transfer to the incubator for 5 minutes.</li>
 <li>Transfer the beads into a Petri dish containing MM, and then into the culture well as previously described. If the beads stick together, which is due to cross-linking between the beads, they can be gently separated with forceps.</li>
</ol>
4. Follicle Imaging and Media Change
<ol>
 <li>Every 2 days, the cultured follicles are imaged using a light microscope and the follicle diameter is measured with the software program ImageJ.</li>
 <li>Every 2 days, half of the growth media (50 &mu;L) is replaced by fresh, pre-equilibrated growth media.</li>
</ol>
5. Follicle Recovery from the 3D Matrix and in vitro Oocyte Maturation (IVM) 
<ol>
 <li>After 8 days of culture, the hydrogels appear clear due to complete degradation of the fibrin component, and the follicles expand to a diameter of 300-400 &mu;m. The remaining alginate is degraded by alginate-lyase, a plant-derived enzyme that specifically degrades alginate and does not affect animal cells.</li>
 <li>Remove growth media from wells containing the beads and add 100 mL of 10 IU/mL alginate lyase in &alpha;MEM . Leave the plate in the incubator for 25-30 minutes.</li>
 <li>Remove follicles from the dissolved beads with a blunt end tip. Transfer first to the outer ring of an IVF dish containing DM, and then into the center well, also containing DM to wash.</li>
 <li>After washing, transfer the follicles to the outer, and then inner ring of an IVF dish containing maturation media. In vitro maturation media (IVM) is composed of &alpha;MEM, 10% FCS, 1.5 IU/mL of hCG, and 5 ng/mL of epidermal growth factor (EGF)</li>
 <li>Transfer to CO2 incubator for 15-16 hours. 
 Examine the follicles for cumulus cells expansion. To remove the cumulus cells from the oocyte, add hyaluronidase solution to final concentration 0.1 mg/mL and incubate in the incubator for 2-3 minutes. Use a 75 mm micropipette tip to shear the cumulus cells from the oocyte by pipetting up and down several times. Determine maturation stage of the oocyte under a light or a dissecting microscope. The possible stages, from least to most mature, are:
 <ul>
 <li>Degenerated. The oocyte is fragmented into two or more pieces.</li>
 <li>Germinal vesicle (GV) stage. The oocyte did not resume meiosis in response to hCG exposure, which is evident by the continued presence of the nucleus of the oocyte (GV).</li>
 <li>Germinal vesicle breakdown (GVBD). The nuclear membrane is absent from the oocyte, but there is no polar body present. Therefore, the oocyte is still in meiosis-I.</li>
 <li>Metaphase-II arrested oocyte (MII). The oocyte resumed meiosis, and is now arrested at metaphase-II. A polar body should be visible on a light or a dissecting microscope. The oocyte will remain at MII unless fertilized in later experiments.</li>
 </ul>
 </li>
</ol>

 
6. Representative Results:
We described a novel encapsulation method of ovarian follicles in a FA-IPN for in vitro culture (Figure 1). Ovarian follicles consist of an oocyte surrounded by several layers of somatic cells. Communication between the multiple cellular compartments is essential for healthy follicle development and oocyte maturation. Follicle encapsulation in a 3D hydrogel supports follicle expansion while maintaining the architecture of the follicle 9,11,12. As follicles develop, their volume expands exponentially and the encapsulating biomaterial should allow this expansion without the development of an inhibiting compressive force. FA-IPN is a network built of two natural materials, where fibrin is proteolytically degradable by plasmin activated by the follicle and alginate is biologically inert 13(Figure 2). During follicle growth, fibrin degradation begins locally near the follicle, and continues until the fibrin is cleared from the hydrogel. The non-degradable alginate component, which remains intact throughout the culture period, supports the 3D structure of the hydrogel.
Follicles were isolated at the secondary stage of development (150-180 &mu;m diameter) and expanded to 400 &mu;m at the antral stage of development in the FA-IPN gels. With hCG stimulation, the cultured follicles can undergo cumulus cell expansion, and oocytes can resume meiosis and progress to metaphase II for fertilization. These results suggest that the encapsulation method and the encapsulating material allowed follicle culture and successful maturation in vitro (Figure 3).
Fibrin degradation around the encapsulated follicles starts on the first day of culture and is completed by day 6. Aprotinin, a soluble plasmin inhibitor, can be used to alter fibrin degradation and to extend mechanical gradient in the encapsulating material (Figure 4). If follicles are cultured with 0.01 TIU/mL aprotinin, the fibrin is degraded slower for the first 4 days. Nonetheless, the follicles can still develop to the antral stage and oocytes remain competent to resume meiosis to metaphase II after aprotinin is removed.A high concentration of aprotinin (0.1 TIU/mL) significantly inhibits fibrin degradation, matrix stiffness prevents follicle expansion and somatic cell invasion into the matrix is observed.
<img src="/files/ftp_upload/2695/2695fig1.jpg" alt="Figure 1" /> 
 Figure 1. Flowchart of follicle development. The ovarian follicle consists of a centrally located oocyte surrounded by one or more layers of somatic cells, which support oocyte development. As follicles develop from secondary to the preovulatory antral stage, the somatic cells surrounding the oocyte proliferate and differentiate, and the oocyte increases in size. in vitro maturation (IVM) is the final step for the follicle culture, when the somatic cells adjacent to the oocyte, termed cumulus cells, expand after hCG stimulation, and the oocyte resumes meiosis and progresses to a metaphase II (MII) stage.
<img src="/files/ftp_upload/2695/2695fig2a.jpg" alt="Figure 2a" /> 
 Figure 2a. Alginate and fibrin-alginate for 3D follicle culture in vitro . (a)Alginate is a natural biomaterial that is suitable for in vitro follicle culture due to its gentle gelation and biochemical characteristics, such as mesh size, controllable rigidity and biological inertness. Alginate is a linear polysaccharide copolymer of &alpha;-L-guluronic acid (G) and &beta;-D-mannuronic acid (M). Areas with repeating G monomers, termed "G-blocks", are cross-linked to form a hydrogel in the presence of divalent calcium ions.
<img src="/files/ftp_upload/2695/2695fig2b.jpg" alt="Figure 2b" /> 
 Figure 2b. (b-i) Small encapsulated follicles experience low compressive force in alginate at the beginning of the culture. However, as the follicle expands the displaced volume in the bead is increasing, which results in greater compressive force in the opposite direction of follicle expansion (b-ii).
 <img src="/files/ftp_upload/2695/2695fig2c.jpg" alt="Figure 2c" /> 
 Figure 2c. The chains of individual polymers are completely entangled in the fibrin-alginate solution prior to cross-linking. Both components of FA-IPN start to cross-link immediately as they are exposed to the mixture of thrombin and calcium.
<img src="/files/ftp_upload/2695/2695fig3a.jpg" alt="Figure 3a" /> 
 Figure 3a. Flowchart for follicle isolation and encapsulation in a FA-IPN. Secondary follicles are isolated from a 16-day old mouse (A-i). The reproductive organs are dissected (A-ii), and isolated follicles are transferred to a dish with maintenance media (A-iii).
<img src="/files/ftp_upload/2695/2695fig3b.jpg" alt="Figure 3b" /> 
 Figure 3b. The drop method for follicle encapsulation in fibrinogen alginate solution (B:i-iv). Two drops of fibrinogen-alginate solution, the rinsing drop (10 &mu;L) and the encapsulation drop (90 &mu;L) are pipetted into the dish (B-i). Next 3-5 follicles are transferred to a rinsing drop (B-ii). After rinsing and media removal, follicles are transferred to the encapsulation drop (B-iii). Each follicle is aspirated with 5 &mu;L fibrinogen-alginate solution with a 10 &mu;L pipette tip and then expelled into thrombin/calcium solution (B-iv).
<img src="/files/ftp_upload/2695/2695fig3c.jpg" alt="Figure 3c" /> 
 Figure 3c. The bead crosslinks for 5 minutes. The parafilm method for follicle encapsulation in fibrinogen alginate solution (C:i-iii). Fibrinogen-alginate solution (7.5 &mu;L) is pipetted on the parafilm coated glass slide and follicles are transferred individually to each drop after rinsing (C-i, ii). Thrombin solution (7.5 &mu;L) is added to each drop (C-iii).
 <img src="/files/ftp_upload/2695/2695fig3d_e.jpg" alt="Figure 3d-e" /> 
 Figure 3d - e. The drops are covered with a second parafilm coated glass slide and the FA-IPN is crosslinked in the incubator for 5 minutes. (D) The encapsulated follicles are transferred to growth media in a 96-well plate. (E) An image of an encapsulated follicle in the FA-IPN (white arrow).
<img src="/files/ftp_upload/2695/2695fig4.jpg" alt="Figure 4" /> 
 Figure 4. Fibrin degradation by the growing follicle. Follicles degrade the fibrin component of the FA-IPN during the culture period, which is demonstrated by a clear circle around the follicle. The follicle's 3D architecture is supported by the remaining alginate. On day 0 (D0) the fibrin is still intact and the matrix around the follicle is cloudy; after 1 day in culture (D1) the clear ring around the follicle appears (white arrow) and the fibrin degradation front continues to radially expand on day 2 (D2) and day 4 (D4) of culture.
<img src="/files/ftp_upload/2695/2695fig5.jpg" alt="Figure 5" /> 
 Figure 5. Images of follicle growth. Representative images ofsecondary follicle growth in the FA-IPN on day 0 (A), 4 (B), 6 (C) and 8 (D) of culture. After 8 days, antral follicles were matured in vitro , and the resulting MII stage oocytes are shown (E, the polar body is shown with black arrow).
<img src="/files/ftp_upload/2695/2695fig6.jpg" alt="Figure 6" /> 
 Figure 6. Fibrin degradation was inhibited by aprotinin. Growing follicles on day 2, 4, 10 and 12 are shown in the first row. Aprotinin at concentrations 0.01 TIU/mL (second row) and 0.1 TIU/mL was added to the culture media on days 0, 2, and 4. Only follicle cultures with 0.01 TIU/mL aprotinin degraded the fibrin after aprotinin removal and reached antral stage. Follicles cultured in 0.1 TIU/mL aprotinin did not grow in the FA-IPN. 
<table border="1">
 <tbody>
 <tr>
 <td>Abbreviation</td>
 <td>Full name</td>
 </tr>
 <tr>
 <td>CO2 incubator</td>
 <td>37&deg;C incubator with 5% CO2</td>
 </tr>
 <tr>
 <td>COC</td>
 <td>Cumulus oocyte complex</td>
 </tr>
 <tr>
 <td>3D</td>
 <td>3 dimensional</td>
 </tr>
 <tr>
 <td>DM</td>
 <td>Dissection media</td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>Epidermal growth factor</td>
 </tr>
 <tr>
 <td>FA-IPN</td>
 <td>Fibrin-alginate interpenetrating network</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Fetal Bovine Serum</td>
 </tr>
 <tr>
 <td>GM</td>
 <td>Growth media</td>
 </tr>
 <tr>
 <td>GV</td>
 <td>Germinal vesicle</td>
 </tr>
 <tr>
 <td>hCG</td>
 <td>Human chorionic gonadotropin</td>
 </tr>
 <tr>
 <td>ITS</td>
 <td>Insulin Transferrin Selenium supplement</td>
 </tr>
 <tr>
 <td>IVF dish</td>
 <td>Center well 60 mm in vitro fertilization dish</td>
 </tr>
 <tr>
 <td>MM</td>
 <td>Maintenance media</td>
 </tr>
 <tr>
 <td>MII stage oocyte</td>
 <td>Metaphase II stage oocyte</td>
 </tr>
 <tr>
 <td>rFSH </td>
 <td>Recombinant Follicular Stimulating Hormone</td>
 </tr>
 <tr>
 <td>TBS</td>
 <td>Tris buffered saline</td>
 </tr>
 <tr>
 <td>TIU</td>
 <td>Trypsin inhibitory units</td>
 </tr>
 </tbody>
</table>
Table 1. Abbreviations

<ol>
	<li>Cortvrindt, R., Smitz, J., VanSteirteghem, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vitro+maturation,+fertilization+and+embryo+development+of+immature+oocytes+from+early+preantral+follicles+from+prepuberal+mice+in+a+simplified+culture+system.">In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system.</a> Human Reproduction. 11, 2656-2666 (1996).</li><li>Smitz, J., Cortvrindt, R., Hu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+combined+with+recombinant+human+chorionic+gonadotrophin+improves+meiotic+progression+in+mouse+follicle-enclosed+oocyte+culture.">Epidermal growth factor combined with recombinant human chorionic gonadotrophin improves meiotic progression in mouse follicle-enclosed oocyte culture.</a> Human Reproduction. 13, 664-669 (1998).</li><li>Xu, M., Banc, A., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+Follicle+Growth+and+Oocyte+Maturation+by+Culture+in+Alginate+Hydrogel+Following+Cryopreservation+of+the+Ovary+or+Individual+Follicles.">Secondary Follicle Growth and Oocyte Maturation by Culture in Alginate Hydrogel Following Cryopreservation of the Ovary or Individual Follicles.</a> Biotechnology and Bioengineering. 103, 378-386 (2009).</li><li>Smitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+achievements+and+future+research+directions+in+ovarian+tissue+culture,+in+vitro+follicle+development+and+transplantation:+implications+for+fertility+preservation.">Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.</a> Human Reproduction Update. 16, 395-414 (2010).</li><li>Shikanov, A., Xu, M., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpenetrating+fibrin-alginate+matrices+for+in+vitro+ovarian+follicle+development.">Interpenetrating fibrin-alginate matrices for in vitro ovarian follicle development.</a> Biomaterials. 30, 5476-5485 (2009).</li><li>West, E. R., Xu, M., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+alginate+hydrogels+and+their+effects+on+in+vitro+follicle+development.">Physical properties of alginate hydrogels and their effects on in vitro follicle development.</a> Biomaterials. 28, 4439-4448 (2007).</li><li>Kreeger, P. K., Fernandes, N. N., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mouse+follicle+development+by+follicle-stimulating+hormone+in+a+three-dimensional+in+vitro+culture+system+is+dependent+on+follicle+stage+and+dose.">Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose.</a> Biology of Reproduction. 73, 942-950 (2005).</li><li>Pangas, S. A., Saudye, H., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+approach+for+the+three-dimensional+culture+of+granulosa+cell-oocyte+complexes.">Novel approach for the three-dimensional culture of granulosa cell-oocyte complexes.</a> Tissue Engineering. 9, 1013-1021 (2003).</li><li>Xu, M., West, E., Shea, L. D., Woodruff, T. 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No conflicts of interest declared.

The presented ovarian follicle encapsulation method in a FA-IPN allows follicle culture in a 3D environment in vitro. A FA-IPN is a dynamic, cell-responsive matrix in which the initial mechanical properties are determined by the combination of both fibrin and alginate. During the culture, the encapsulated follicle activates proteases that degrade only one component of the IPN, the fibrin, which results in a gradually decreasing gel rigidity that is contributed solely by the remaining alginate at the end of the c...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fetuin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>F3385</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen, Gibco</td>
<td>10082-139</td>
<td>&nbsp;</td>
<tr>
<td>Aprotinin</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10236624001</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Wako</td>
<td>039-00475</td>
<td>40 mM</td>
</tr>
<tr>
<td>EGF</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A412</td>
<td>&nbsp;</td>
<tr>
<td>rFSH</td>
<td>&nbsp;</td>
<td>A.F. Parlow, National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>hCG</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>CG-5</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1603</td>
<td>&nbsp;</td>
<tr>
<td>ITS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I1884-1VL</td>
<td>&nbsp;</td>
<tr>
<td>L-15</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11415</td>
<td>&nbsp;</td>
<tr>
<td>&alpha;MEM+Gluta MAX</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>32561</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
<tr>
<td>TBS</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28379</td>
<td>&nbsp;</td>
<tr>
<td>Tisseel Fibrin kit</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>921030</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Alginate</td>
<td>&nbsp;</td>
<td>FMC BioPolymers</td>
<td>LF200DL</td>
<td>Mw 418kDa</td>
</tr>		</tbody>
		</table>
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a method for ovarian follicle encapsulation and culture in a proteolytically degradable 3 dimensional system

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

Watch this Scientific Journal Video about A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System at JoVE.com

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

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Research

JoVE Journal

Bioengineering

24.2K ビュー.  Northwestern University. 3Dフィブリン-アルギン酸相互貫入ネットワークにおける卵胞のカプセル化のための新しい方法が記載されている。このシステムは、成熟した卵母細胞を生成するために未熟な卵胞の発達をサポートするためにタンパク質分解と構造的な支持を兼ね備えています。このメソッドは、拡張を制限することなく、細胞間接触を維持するために培養細胞の集合体?...

ビデオ: A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...