Name: generating spheroids from various chondrocytes using low-adhesive conditions under gravity and homemade mini-bioreactors
Uploaded: 2025-01-31T13:20:00.000+00:00
Duration: 9 min 3 s
Description: Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...

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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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><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&#039;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 &amp;mu;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 &amp;mu;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&amp;nbsp;</td><td>PanEco</td><td>P011-2</td><td>0.01%</td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole)</td><td>Sigma-Aldrich</td><td>D9542-5MG</td><td>1 &amp;mu;g/mL</td></tr><tr><td>DMEM medium w/o glutamine</td><td>PanEco</td><td>?420?</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>10270106</td><td>10%</td></tr><tr><td>Hanks&#039; solution&amp;nbsp;</td><td>PanEco</td><td>?020?</td><td></td></tr><tr><td>Hybris 8 medium</td><td>PanEco</td><td>?780?/?780</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium solution</td><td>PanEco</td><td>?065</td><td>1x solution has the following concentrations: Insulin: 10 &amp;micro;g/mL; Transferrin: 5.5 &amp;micro;g/mL; Selenium 5 ng/mL</td></tr><tr><td>L-alanyl-L-glutamine&amp;nbsp;</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 &amp;mu;g/mL</td></tr><tr><td>Penicillin-Streptomycin solution</td><td>PanEco</td><td>?063?</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>?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-&amp;beta;1&amp;nbsp;</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

317 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

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

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

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

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

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

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

Biochemistry

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

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 ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix.&#160;This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 &#176;C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization.&#160;The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

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

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Chapters in this video

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

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

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

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

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production