The research was supported with allocation #22-15-00250 by the Russian Science Foundation.

&#8203;The study was reviewed and approved by the Ethics Committee of the LOPUKHIN FRCC PCM (protocol No. 2019/02 of April 9, 2019). All donor samples were obtained in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from all participants and/or their legal guardians.
NOTE: Maintain sterile technique throughout the protocol. Warm all culture media and solutions to 37 &#176;C before applying them to cells or spheroids. Cultivate cells in a CO2 incubator at 37 &#176;C, with 5% CO2 upon 80% humidity. The protocol scheme is shown in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67481/67481fig1.jpg" /> 
Figure 1: Protocol for producing chondrospheres from iPSC line IPSRG4S. (A) Initially, iPSCs are cultured in pluripotent stem cell media until 80% confluency is reached. (B) To induce differentiation towards the chondrocytic linage, iPSCs are then cultured in medium A for 2 days. The medium is subsequently replaced with a formulation lacking Chir 99021 and Rho kinase inhibitor, and cells are cultured for an additional 6 days with medium changes every 2 days. (C) The cells are then transferred to medium B to promote chondrogenesis for the next 10 days. (D) Once a sufficient cell number is achieved, the process advances to the spheroids production stage. (E) The cells are aggregated into spheroids in low-adhesive conditions under gravity. (F) The resulting spheroids are cultured in mini-bioreactors with medium B. <a href="https://www.jove.com/files/ftp_upload/67481/67481fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Transformation of Petri dishes into mini-bioreactors
NOTE: Details in a previous article5.
<ol>
	<li>Slice centrifuge tubes into 7-8 mm high rings using sterile autoclave scissors (autoclave settings: 121 &#176;C (250 &#176;F) at 15 psi, 30 min, and post-autoclave drying for 20 min).</li>
	<li>Crush low-adhesion, untreated, or microbiological Petri dishes into small pieces. Dissolve approximately 1 g of plastic particles in 10 mL of chloroform overnight to create a liquid plastic solution. Conduct this procedure in a fume hood.</li>
	<li>Verify that the liquid plastic is sufficiently viscous for pipetting. Ensure that droplets maintain a spherical shape rather than spreading on the surface. If the solution is too thin, add more plastic particles, usually requiring 1-3 g. If it is too thick, add additional chloroform, usually requiring up to 5 mL.</li>
	<li>Form a plastic knob at the center of a sterile, ultra-low adhesion 60 mm Petri dish using one of the two effective methods outlined below.
	<ol>
		<li>Option 1: Place the autoclaved plastic ring in the center of the Petri dish, then apply liquid plastic inside the ring; one drop will require about 100-200 &#181;L drops of liquid plastic.</li>
		<li>Option 2: Apply the same number of drops of liquid plastic directly into the center of the Petri dish without using the plastic ring.</li>
	</ol>
	</li>
	<li>Allow the dishes to dry uncovered for 2-3 h in a laminar flow hood. After drying, expose the dishes to ultraviolet radiation for 20-30 min.</li>
</ol>
2. Treatment of culture dishes with gelatin
<ol>
	<li>Prepare a 0.1% gelatin solution by dissolving powdered gelatin in distilled water at 95 &#176;C. Pass the solution sequentially through 0.45 &#181;m and 0.25 &#181;m membrane filters, then store the filtrate at 4 &#176;C.</li>
	<li>Immediately before chondrocyte reseeding, apply the 3 mL of gelatin solution to the 25 cm2 flask (up to 15 g of cartilage tissue per T25 flask is recommended). Incubate at room temperature (RT) for 30 min,remove the gelatin, rinse twice with PBS, and proceed with cell seeding.</li>
</ol>
3. Isolation and cultivation of chondrocytes
NOTE: Chondrocytes are isolated from non-weight-bearing cartilage regions from patients undergoing total knee replacement. Harvest all specimens under sterile conditions and store them in sterile tubes filled with DMEM containing 200 U/mL penicillin/streptomycin solution. Store cartilage tissue in a refrigerator at 2-8 &#176;C.
<ol>
	<li>Begin enzymatic treatment within 2 days of cartilage harvest. Wash cartilage pieces in Hanks&#39; solutionusing 60 mm Petri dishes, then cut cartilage into fragments of approximately 1-2 mm in diameter using autoclaved scissors.</li>
	<li>Transfer cartilage pieces into 15 mL tubes and digest them in 0.01% collagenase type II solution in DMEM, approximately 1 mL per 1 g of cartilage, within 8-12 h in a CO2 incubator set to 37 &#176;C with 5% CO2, 100% humidity, under continuous agitation on an orbital shaker at 150-200 rpm.</li>
	<li>After digestion, wash the cartilage fragments by adding DMEM to a 15 mL tube, then centrifuge at 300 &#215; g for 10 min. Resuspend the fragments in a chondrocyte culture medium (Supplementary Table 1) and seed them onto an adhesive T25 flask pre-coated with 0.1% gelatin solution.</li>
	<li>Passage chondrocytes when a confluent density of 80% is achieved.
	<ol>
		<li>For passaging, wash chondrocytes with Hanks&#39; solution, then incubate cells with 0.25% trypsin solution at 37 &#176;C for 5 min.</li>
		<li>To inactivate trypsin, add an equal volume of DMEM + 10% FBS. Resuspend chondrocytes in fresh culture medium, then distribute them at a 1:3 ratio, seeding 20,000-30,000 cells per 1 cm2.</li>
		<li>Place the culture flask in an incubator at 37 &#176;C with 5% CO2 and 100% humidity.</li>
	</ol>
	</li>
</ol>
4. Cultivation of pluripotent stem cells
NOTE: In collaboration with the Stem Cell Laboratory of the Molecular Brain Research Group, Department of Neurobiology, A.I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland, an iPSCs line IPSRG4S, was successfully generated in a previous study8.
<ol>
	<li>Cultivate iPSCs in 6-well plates pre-coated with a matrix containing extracellular proteins ( Figure 1A). Use a medium for pluripotent stem cells (Supplementary Table 1).</li>
	<li>Passage cells at 80% confluency, maintaining a 1:4 ratio (50,000-75,000 cells per 1 cm2). Approximately 2-3 h before detachment, transfer iPSCs to the medium for pluripotent stem cells containing Rho kinase inhibitor Y27632 at a final concentration 1 &#181;M for 1 day.</li>
	<li>Wash iPSCs with Hanks&#39; solution and incubate at 37 &#176;C with 0.05% trypsin solution. To inactivate trypsin, add an equal volume of DMEM + 10% FBS. Centrifuge the cell suspension at 200 x g for 5 min in 15 mL tubes.</li>
	<li>Discard the supernatant and resuspend the cells in a fresh medium for pluripotent stem cells. To enhance cell viability, add Rho kinase inhibitor Y27632 to the medium at a final concentration of 1 &#181;M for 1 day. Place the culture plate in a CO2 incubator set to 37 &#176;C with 5% CO2 and 100% humidity.</li>
</ol>
5. Chondrogenic differentiation of iPSCs
<ol>
	<li>Initiate differentiation towards the chondrocytic lineage by culturing cells in Medium A (Supplementary Table 1) for the first 2 days. On day 3, replace Medium A with a solution that excludes Chir 99021 and Rho kinase inhibitor while keeping all other components unchanged. Change the medium every 2 days from days 3 to 9 (Figure 1B).</li>
	<li>On day 10, transfer chondrocyte-like cells to Medium B (Supplementary Table 1), formulated to facilitate chondrogenesis. Change the medium every 2 days for 10 days (Figure 1C).</li>
	<li>Prepare a 96-well cell suspension culture plate in advance by coating each well with 1.5% agarose in distilled water. Melt the agarose in a microwave until boiling (about 60-90 s at 700 W). Once agarose is liquefied, add 75 &#181;L of agarose to each well and allow it to harden at RT (approximately 15 min).</li>
	<li>Add 150 &#181;L of DMEM medium to each well and incubate the plate in a CO2 incubator for at least 12 h. 
	NOTE: Prepared plates can be stored for up to 1 month in a CO2 incubator at 37 &#176;C if the medium in each well is replenished to compensate for evaporation.</li>
	<li>On day 12, after observing phenotypical changes associated with chondrogenesis, initiate spheroids formation using the generated chondrocyte-like cells. 
	NOTE: At this stage, it is advisable to analyze the expression of key chondrocytic markers with immunocytochemistry (ICC) and quantitative polymerase chain reaction (qPCR) using the 2-&#916;&#916;CT method9 to confirm successful chondrogenic differentiation of iPSCs. During passaging from 6-well plate to a 75 cm2 cell culture flask, a small portion of cells can be seeded into a 48-well plate, matching the number of markers to be analyzed. Perform ICC analysis on these samples to verify gene expression.</li>
</ol>
6. Formation of spheroids from chondrocyte-like iPSC-derived cells
<ol>
	<li>After obtaining chondrocyte-like derivatives, passage cells at 80% confluency. Detach cells from the 6-well plates using a 0.05% trypsin solution. To inactivate trypsin, add an equal volume of DMEM + 10% FBS, and then transfer cells to a 15 mL tube and centrifuge at 200 x g for 5 min.</li>
	<li>Remove the supernatant, resuspend the cells in 1 mL of Medium B, and transfer to a 75 cm2 cell culture flask pre-coated with 0.1% gelatin solution (Figure 1D). Once cells reach 80% confluency as a monolayer, detach cells again with trypsin and resuspend in Medium B as described.</li>
	<li>Transfer cells into a 96-well plate coated with 1.5% agarose at a density of 100,000 cells per well. Cultivate in Medium B, adding 150 &#181;L of complete medium per well (Figure 1D, 3A).</li>
	<li>Maintain cells in the 96-well plate coated with 1.5% agarose for a minimum of 1 day and a maximum of 3 days until spheroids are formed (Figure 2B). Transfer spheroids from the wells using a 1 mL pipette tip with a trimmed end, then transfer to a 15 mL tube. Allow spheroids to settle for 2-3 min and remove the supernatant.</li>
	<li>Immerse the spheroids in freshly thawed, undiluted basement membrane matrix solution maintained at 4 &#176;C. After 30 min, collect spheroids by passive sedimentation in a 15 mL tube or by gentle centrifugation at 100 x g for 1 min. 
	NOTE: Matrix should be thawed overnight on ice in a 2-8 &#176;C refrigerator. After the first thaw, single-use aliquots of the matrix are made to avoid repeated freeze-thaw cycles. Polypropylene or other freezer-compatible tubes are used and stored at -70 &#176;C or -20 &#176;C. Be watchful of the expiration date of the batch/lot, which is 2 years from the manufacture date.</li>
	<li>Transfer spheroids into mini-bioreactors and add 6 mL of Medium B. Place the mini-bioreactor on an orbital shaker inside a CO2 incubator set to 37 &#176;C with 5% CO2 and 100% humidity and an agitation speed of 70-75 rpm (Figure 1F).</li>
	<li>Change the medium weekly or as required by medium depletion or color change of the acid-base indicator. To do this, allow spheroids to settle by gravity in a 15 mL tube, and then carefully remove the supernatant. Add fresh Medium B to the tube, and transfer spheroids back to the mini-bioreactors.</li>
</ol>

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substitutes from cells with induced pluripotency.</a> Med Extrem Situat. 4, 30-41 (2022).</li><li>Rodr&#237;guez Ruiz, A., 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=Cartilage+from+human-induced+pluripotent+stem+cells:+comparison+with+neo-cartilage+from+chondrocytes+and+bone+marrow+mesenchymal+stromal+cells.">Cartilage from human-induced pluripotent stem cells: comparison with neo-cartilage from chondrocytes and bone marrow mesenchymal stromal cells.</a> Cell Tissue Res. 386, 309-320 (2021).</li><li>Eremeev, A., 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=Brain+organoid+generation+from+induced+pluripotent+stem+cells+in+homemade+mini+bioreactors.">Brain organoid generation from induced pluripotent stem cells in homemade mini bioreactors.</a> J Vis Exp. 178, e62987 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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

iPSCs represent a transformative tool in regenerative medicine, offering the potential to generate patient-specific chondrocytes for cartilage repair. Current protocols leverage directed differentiation through mesodermal pathways, with key signaling molecules like TGF-&#946; and BMP-2 promoting chondrocytic lineage commitment. These methods aim to replicate embryonic cartilage development, enabling the production of extracellular matrix components such as collagen type II and aggrecan, essential for functional cartilage...

The prevalence of joint disease leads to significant economic burdens due to the increasing number of disabled patients and the costs associated with their care. Hyaline cartilage is a connective avascular tissue with limited regenerative potential1. Prolonged use of certain non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and chemotherapy or radiotherapy can further diminish cartilage&#39;s regenerative capacity, nearly eliminating its ability to heal2. This makes it challenging to obtain autologous cartilage cells for cellular therapy.
The technology of three-dimensional (3D) cell cultivation, including chondrocytes, has long been recognized as an emerging research area with significant potential. These 3D structures are being studied for applications in both fundamental biological research and regenerative medicine. Spheroids derived from autologous chondrocytes hold therapeutic promise for addressing cartilage tissue degeneration, a condition currently receiving considerable global attention3.
Spheroids derived from chondrocytes differentiated from iPSCs represent a promising alternative to primary chondrocytes, offering significant advantages for cartilage repair. iPSCs provide an almost limitless capacity for self-renewal and possess a broad differentiation potential, which allows for the production of chondrocytes in quantities sufficient for clinical applications without invasive procedures for cell collection4. Moreover, transitioning from traditional two-dimensional (2D) chondrocyte cultures to 3D culture systems, such as chondrospheres, further enhances the viability and functionality of these cells by creating a more physiologically relevant environment. Studies show that chondrocytes cultured in 3D spheroids better maintain their phenotype, displaying lower rates of dedifferentiation and higher expression levels of key hyaline cartilage markers, such as collagen type II and aggrecan5.
Despite the potential of iPSC-derived chondrospheres, standardized protocols for generating high-quality chondrospheres remain limited. Variability in protocols across studies often leads to inconsistencies in chondrocyte quality and extracellular matrix composition, affecting their effectiveness for therapeutic use6. Here, a refined protocol is presented that standardizes the generation of spheroids from iPSC-derived chondrocytes using affordable, custom-made mini-bioreactors. The mini-bioreactor culture phase is essential, as it provides a controlled, low-adhesion setting that optimizes nutrient distribution, ECM maturation, and spheroid compaction7. This phase promotes robust expression of essential chondrocyte markers, including collagen type II, aggrecan, and SOX9, along with an ECM composition that closely resembles native cartilage. Regular media changes and careful control of environmental conditions -- temperature, CO2, and rotation speed -- further ensure the viability and maturation of the chondrocyte spheroids. This protocol has been optimized to create high-quality chondrospheres with strong expression of hyaline cartilage markers in a cost-effective and scalable manner, suitable for research and clinical applications in cartilage repair.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12 Eagle&#39;s medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Aggrecan Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>AHP0022</td>
			<td>Host: Mouse; Dilution: 1/500</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td>50 &#956;g/mL</td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td>1x or 2%</td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Serva</td>
			<td>28625</td>
			<td>90 mM</td>
		</tr>
		<tr>
			<td>BMP2</td>
			<td>Miltenyi biotec</td>
			<td>130-110-922</td>
			<td>10 ng/mL</td>
		</tr>
		<tr>
			<td>Chir 99021</td>
			<td>Miltenyi biotec</td>
			<td>130-103-926</td>
			<td>10 &#956;M</td>
		</tr>
		<tr>
			<td>COL1A1 (E6A8E) Monoclonal antibody</td>
			<td>CellSignalling</td>
			<td>39952</td>
			<td>Host: Rabbit; Dilution: 1/800</td>
		</tr>
		<tr>
			<td>COL2A1 (M2139) Monoclonal antibody</td>
			<td>Santa Cruz</td>
			<td>sc-52658</td>
			<td>Host: Mouse; Dilution: 1/50</td>
		</tr>
		<tr>
			<td>Collagenase type II solution&#160;</td>
			<td>PanEco</td>
			<td>P011-2</td>
			<td>0.01%</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-5MG</td>
			<td>1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>DMEM medium w/o glutamine</td>
			<td>PanEco</td>
			<td>&#1057;420&#1087;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270106</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>Hanks&#39; solution&#160;</td>
			<td>PanEco</td>
			<td>&#1056;020&#1087;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybris 8 medium</td>
			<td>PanEco</td>
			<td>&#1057;780&#1045;/&#1060;780</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium solution</td>
			<td>PanEco</td>
			<td>&#1060;065</td>
			<td>1x solution has the following concentrations: Insulin: 10 &#181;g/mL; Transferrin: 5.5 &#181;g/mL; Selenium 5 ng/mL</td>
		</tr>
		<tr>
			<td>L-alanyl-L-glutamine&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050038</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Matrigel Matrix</td>
			<td>BD</td>
			<td>354277</td>
			<td>300 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>PanEco</td>
			<td>&#1040;063&#1087;</td>
			<td>100 U/mL</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Miltenyi biotec</td>
			<td>130-117-339</td>
			<td>10 nM</td>
		</tr>
		<tr>
			<td>Rho kinase inhibitor Y27632</td>
			<td>Miltenyi biotec</td>
			<td>130-103-922&#160;</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Secondary Antibody Goat anti-Mouse IgG (H+L) Cross-Adsorbed, Alexa Fluor 555</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>A21422</td>
			<td>Host: Goat; Dilution: 1/500</td>
		</tr>
		<tr>
			<td>Secondary Antibody Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed, Alexa Fluor Plus 555</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>&#1040;32732</td>
			<td>Host: Goat; Dilution: 1/500</td>
		</tr>
		<tr>
			<td>Sox9 (D8G8H) Monoclonal antibody</td>
			<td>CellSignalling</td>
			<td>82630</td>
			<td>Host: Rb; Dilution: 1/400</td>
		</tr>
		<tr>
			<td>TeSR-1 medium</td>
			<td>STEMCELL technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946;1&#160;</td>
			<td>Miltenyi biotec</td>
			<td>130-095-067</td>
			<td>10 ng/mL</td>
		</tr>
	</tbody>
</table>

generating spheroids from various chondrocytes using low-adhesive conditions under gravity and homemade mini-bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

The outlined protocol is illustrated in Figure 1. This methodology employs two distinct culture media to drive the differentiation of iPSCs into chondrocyte spheroids over a minimum duration of 1 month (Figure 2). The differentiation process is initiated when iPSCs achieve 75%-90% confluency (Figure 1B). Early indicators of chondrogenic differentiation emerge around days 9-10 of cultivation in medium...

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...

generating-spheroids-from-various-chondrocytes-using-low-adhesive

Research

JoVE Journal

Bioengineering

222 Views.  Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency. This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Video: Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

Lab-on-a-CD Platform for Generating Multicellular Three-dimensional Spheroids

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the living body than two-dimensional cell culture. In this study, we fabricated an electrical motor-driven lab-on-a-CD (compact disc) platform, called a centrifugal microfluidic-based spheroid (CMS) culture system, to create three-dimensional (3D) cell spheroids implementing high centrifugal force. This device can vary rotation speeds to generate gravity conditions from 1 x g to 521 x g. The CMS system is 6 cm in diameter, has one hundred 400 &#956;m microwells, and is made by molding with polydimethylsiloxane in a polycarbonate mold premade by a computer numerical control machine. A barrier wall at the channel entrance of the CMS system uses centrifugal force to spread cells evenly inside the chip. At the end of the channel, there is a slide region that allows the cells to enter the microwells. As a demonstration, spheroids were generated by monoculture and coculture of human adipose-derived stem cells and human lung fibroblasts under high gravity conditions using the system. The CMS system used a simple operation scheme to produce coculture spheroids of various structures of concentric, Janus, and sandwich. The CMS system will be useful in cell biology and tissue engineering studies that require spheroids and organoid culture of single or multiple cell types.

We present a motor-powered centrifugal microfluidic device that can cultivate cell spheroids. Using this device, spheroids of single or multiple cell types could be easily cocultured under high gravity conditions.

A three-dimensional spheroid cell culture can obtain more useful results in cell experiments because it can better simulate cell microenvironments of the ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...