This research is supported by a collaboration with FUKOKU, CO., Ltd. Takao Yoshida from FUKOKU, CO., Ltd. provided the idea of the O-shaped culture bag. Takamasa Sato from FUKOKU, CO., Ltd. supported this research in terms of a computational simulation for developing the O-shaped culture bag. We would like to appreciate the corresponding author&#39;s current affiliation, Osaka University, for allowing us to work on the publication.

1. Preparation of the Cells and Materials
<ol>
	<li>Cultivate HEK293 cells in Dulbecco&#39;s modified Eagle medium with a 10% fetal bovine serum (FBS) and 1% non-essential amino acids. Adjust the pH of the culture medium to 6.8 - 7.6.</li>
	<li>After culturing for 5 to 7 d, dissociate the cells with a 0.25% trypsin-ethylenediaminetetraacetic acid solution (trypsin-EDTA) and reseed the cells at 5,000-10,000 cells/cm2.</li>
</ol>
2. Seeding the Cells in an O-shaped Bag
<ol>
	<li>Dissociate the cells as described in step 1.2 and resuspend them in 2 - 5 mL of culture medium.</li>
	<li>Filter the cell suspension through a cell strainer (40 &#181;m) to collect a single cell suspension.</li>
	<li>Count the number of cells by trypan blue staining and automatic cell counter, and then prepare 20 mL of the cell suspension (2.0 x 105 cells/mL).</li>
	<li>Connect the inlet of the O-shaped bag (Figure 1a) to a clamped 50 mL syringe without a plunger.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57922/57922fig1.jpg" /> 
Figure 1: Schematic images and pictures of O-shaped vessels. (a1) This is a schematic image of an O-shaped bag. (a2) This is a picture of an O-shaped bag. (b1) This is a schematic image of an O-shaped dish. (b2) This is a picture of an O-shaped dish. The outer and inner diameter of the O-shaped bag are 90 mm and 20 mm, respectively. <a href="https://www.jove.com/files/ftp_upload/57922/57922fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Pipette the prepared cell suspension into the O-shaped bag through the clamped syringe.</li>
	<li>Replace the first clamped syringe with a clean syringe. Add 55 mL of clean air through the clean syringe to expand the bag completely.</li>
	<li>Clamp the inlet tube and then close the inlet. Finally, remove the clamp from the tube.</li>
	<li>Incubate the cells in the O-shaped bag by shaking them at 45 rpm in conditions of 37 &#176;C and 5% CO2.</li>
</ol>
3. Medium Change (optional)
<ol>
	<li>Transfer the cell suspension into a 50 mL tube through the inlet.</li>
	<li>Centrifuge it for 2 min at 200 x g at room temperature, and then aspirate the supernatant.</li>
	<li>Add 20 mL of culture medium and resuspend the cells.</li>
	<li>Add the resuspended cells to the O-shaped bag by following steps 2.4 - 2.7.</li>
	<li>Incubate the cells in the O-shaped bag by shaking them at 45 rpm in conditions of 37 &#176;C and 5% CO2.</li>
</ol>
4. Collecting the Cells from the Bag
<ol>
	<li>Transfer the cell suspension into a 50 mL tube through the inlet.</li>
	<li>Wash the inside of the bag with 20 mL of calcium/magnesium-free phosphate buffered saline [PBS(-), pH = 7.4 - 7.6] and then drain the contents into a tube to collect the remaining cells from the bag.</li>
	<li>Centrifuge the collected cell suspension for 3 min at 200 x g at room temperature, and then aspirate the supernatant.</li>
	<li>Add 10 mL of PBS(-) and wash the aggregates.</li>
	<li>Centrifuge them for 3 min at 200 x g at room temperature, and then aspirate the supernatant to collect the aggregates.</li>
	<li>Add 4 mL of PBS and 1 mL of trypsin and incubate them with the aggregates for 10 min at 37 &#176;C to dissociate the cells for counting (optional).</li>
</ol>
5. Preparation of an O-shaped Dish (optional)
NOTE: See Figure 1b for a schematic image of the O-shaped dish.
<ol>
	<li>Cut out the bottom of a 60 mm or 35 mm dish with a hot knife and utilize it as an inner dish.</li>
	<li>Put the 60 mm dish upside-down on the center of a 100 mm dish. 
	NOTE: A guide sheet can help to decide the position of the 60 mm dish.</li>
	<li>If required, put viscosity-adjusted cyclohexanone on the commissure from the inside of 60 mm dish.</li>
	<li>Dry the O-shaped dish for a few days and sterilize it by gamma ray or ethylene oxide gas.</li>
</ol>

<ol>
	<li>Mihara, Y., 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=Production+of+pancreatic+progenitor+cells+from+human+induced+pluripotent+stem+cells+using+a+three-dimensional+suspension+bioreactor+system.">Production of pancreatic progenitor cells from human induced pluripotent stem cells using a three-dimensional suspension bioreactor system.</a> Journal of Tissue Engineering and Regenerative Medicine. 11 (11), 3193-3201 (2017).</li><li>Raven, N., 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=Scaled-up+manufacturing+of+recombinant+antibodies+produced+by+plant+cells+in+a+200-L+orbitally-shaken+disposable+bioreactor.">Scaled-up manufacturing of recombinant antibodies produced by plant cells in a 200-L orbitally-shaken disposable bioreactor.</a> Biotechnology and Bioengineering. 112 (2), 308-321 (2015).</li><li>Han, Y., 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=Cultivation+of+recombinant+chinese+hamster+ovary+cells+grown+as+suspended+aggregates+in+stirred+vessels.">Cultivation of recombinant chinese hamster ovary cells grown as suspended aggregates in stirred vessels.</a> Journal of Bioscience and Bioengineering. 102 (5), 430-435 (2006).</li><li>Portolano, N., 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=Recombinant+Protein+Expression+for+Structural+Biology+in+HEK+293F+Suspension+Cells:+A+Novel+and+Accessible+Approach.">Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach.</a> Journal of Visualized Experiments. (92), e51897 (2014).</li><li>Himmelfarb, P., Thayer, P. S., Martin, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spin+filter+culture:+the+propagation+of+mammalian+cells+in+suspension.">Spin filter culture: the propagation of mammalian cells in suspension.</a> Science. 164 (3879), 555-557 (1969).</li><li>Kropp, C., Massai, D., Zweigerdt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+challenges+in+large-scale+expansion+of+human+pluripotent+stem+cells.">Progress and challenges in large-scale expansion of human pluripotent stem cells.</a> Process Biochemistry. 59, 244-254 (2017).</li><li>Singh, H., Mok, P., Balakrishnan, T., Rahmat, S. N. B., Zweigerdt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-scaling+single+cell-inoculated+suspension+culture+of+human+embryonic+stem+cells.">Up-scaling single cell-inoculated suspension culture of human embryonic stem cells.</a> Stem Cell Research. 4 (3), 165-179 (2010).</li><li>Horiguchi, I., Sakai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+replacement+with+albumin-associated+lipids+prevents+excess+aggregation+and+enhances+growth+of+induced+pluripotent+stem+cells+in+suspension+culture.">Serum replacement with albumin-associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture.</a> Biotechnology Progress. 32 (4), 1009-1016 (2016).</li><li>Sutherland, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygenation+and+differentiation+in+multicellular+spheroids+of+human+colon+carcinoma.">Oxygenation and differentiation in multicellular spheroids of human colon carcinoma.</a> Cancer Research. 46 (10), 5320-5329 (1986).</li><li>Hang, H., 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=Computational+fluid+dynamics+modeling+of+an+inverted+frustoconical+shaking+bioreactor+for+mammalian+cell+suspension+culture.">Computational fluid dynamics modeling of an inverted frustoconical shaking bioreactor for mammalian cell suspension culture.</a> Biotechnology and Bioprocess Engineering. 16 (3), 567-575 (2011).</li><li>Einstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cause+of+the+formation+of+meanders+in+the+courses+of+rivers+annd+of+the+so-called+bear's+law.">The cause of the formation of meanders in the courses of rivers annd of the so-called bear's law.</a> Die Naturwissenschaften. 14, (1926).</li><li>Tandon, A., Dartmouth, U., Marshall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Einstein's+tea+leaves+and+pressure+systems+in+the+atmosphere.">Einstein's tea leaves and pressure systems in the atmosphere.</a> The Physics Teacher. 48, 292-295 (2010).</li><li>Miyamoto, D., Nakazawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+mouse+iPS+cells+is+dependent+on+embryoid+body+size+in+microwell+chip+culture.">Differentiation of mouse iPS cells is dependent on embryoid body size in microwell chip culture.</a> Journal of Bioscience and Bioengineering. 122 (4), 507-512 (2016).</li><li>Olmer, R., 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=Suspension+culture+of+human+pluripotent+stem+cells+in+cotrolled,+stirred+bioreactors.">Suspension culture of human pluripotent stem cells in cotrolled, stirred bioreactors.</a> Tissue Engineering Part C: Methods. 18 (10), 772-784 (2012).</li><li>Singh, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disposable+bioreactor+for+cell+culture+using+wave-induced+agitation.">Disposable bioreactor for cell culture using wave-induced agitation.</a> Cytotechnology. 30, 149-158 (1999).</li></ol>

The authors have nothing to disclose.

In this study, we developed O-shaped vessels and performed an HEK293 suspension culture in them for a uniform aggregates formation and expansion. In the conventional culture dish, an orbital shaking culture produced two different diameters of aggregates, whereas we observed uniform aggregates in the O-shaped vessels (Figure 1). According to the observation of the distribution of stained beads in the orbital shaking conditions, beads gather in the center-bottom of a conventional culture dish. That gathering presumably caused the difference in cell density leading to various sizes of aggregates. Alternatively, beads were distributed in the O-shaped dish which does not have the center-bottom region (Supplemental Figure 1). This distribution probably causes the uniformly-sized aggregates in O-shaped vessels. Another&#8212;widely used&#8212;approach to producing uniform aggregates is the culturing in microwells, but this approach has some problems, such as in supplying a culture medium without aggregates dropping out of the microwells13. In the case of O-shaped vessels, uniform aggregates can be produced in a simple suspension culture.
The orbital shaking culture is a popular culture system utilized for mammalian cells. There are various culture methods for the mass production of mammalian cells, such as the stirred tank bioreactor14, wave-motioned bags15, and rotating bottles. Orbital shaking cultures do not include an inner impeller for stirring the medium, unlike the stirred tank bioreactor does. This feature is similar to the wave-motioned bags and rotating bottles. These impeller-free culture systems can avoid cellular damage from the shear force surrounding the impellers and realize low shear stress in suspension culture. Especially, orbital shaking culture systems are effective for the mass production of sensitive cells such as mammalian cells because of their high scalability and low shear stress.
O-shaped vessels can improve the remaining problem of orbital shaking culture in the formation of aggregates. In orbital shaking culture systems, floating cells migrate to the center and bottom of the vessel, which is known as the &#34;Einstein&#39;s tea leaf paradox&#34;11. This migration causes the inhomogeneous aggregation and non-uniform aggregate production in conventional orbital shaking vessels. In this study, O-shaped vessels prevented the concentration of cells into the center-bottom of the vessels, which is speculated as the reason of uniform aggregation in orbital shaking O-shaped vessels.
Histological analyses showed that the aggregates in the conventional dish contained denucleated cells (Figure 2a). In contrast, denucleated cells did not appear in the aggregates from O-shaped vessels (Figure 2b and 2c). It is possible that these denucleated cells were caused by a shortage of substrates such as glucose, glutamine, and oxygen9. According to the size measurement, aggregates in the O-shaped vessels had homogeneous diameters lower than 400 &#181;m. In contrast, in a conventional dish, some aggregates had a diameter larger than 400 &#181;m, and these aggregates included denucleated cells. This result suggests that creating homogeneous-sized aggregates in O-shaped vessels is effective in controlling the quality of aggregates. In addition, it is also speculated that the oxygenation through the gas-permeable polyethylene film prevented the appearance of denucleated cells in the O-shaped bag.
These experiments showed the possibility of these O-shaped vessels as a simple system for producing uniform aggregates. Although other culture bags for suspension culture have been developed15, those culture bags are square-shaped, which prevents the culture from getting effectively mixed in orbital shaking. The bag in this study has a novel round shape suitable for orbital shaking to produce aggregates with a homogeneous size. This characteristic of vessels is important for controlling the conditions and the high reproducibility of cells in mass production. The possible application of the O-shaped vessel is widespread. It can be used when producing recombinant proteins from cells and for regenerative medicine when dealing with stem cells.
In conclusion, we developed a novel O-shaped bag suitable for producing uniform cell aggregates with an orbital shaking culture. The bag shows possibilities for various biomedical applications such as in regenerative medicine.

Suspension cell culture plays an important role in the cell production for regenerative medicine1 and recombinant protein production2 because it is easy to scale up and achieve high cell densities, sometimes up to more than 107 cells/mL5. Chinese hamster ovary (CHO) cells3 and human embryonic kidney cells 293 (HEK293)4 have been grown in suspension culture, and human pluripotent stem cells (hPSCs) have recently been grown in suspension culture systems for regenerative therapies1,6,7. The use of suspension cultures is expected to increase in the future.
In suspension culture, some cell lines cannot grow as single cells and must, therefore, form aggregates. For example, hPSCs cannot survive in suspension conditions without forming aggregates. However, this requirement for aggregated growth presents difficulties for uniform suspension cultures. One difficulty is the formation of uniform aggregates in the early period of the culture, which determines the efficiency of the suspension culture8. Another difficulty is the mass transfer of nutrients into aggregates. In particular, the oxygen supply limits the maximum size of aggregates, and a poor oxygen supply causes necrosis in the center of aggregates9. Consequently, suspension cultures of cell aggregates are more difficult to obtain than conventional suspension cultures. Nevertheless, suspension cultures are crucial for biomedical and biotechnology applications.
Orbital shaking vessels are one of the simplest suspension culture systems that achieve culture medium mixtures without impeller agitation. Shear stress from the impeller and the dynamic medium flow is the major problem of suspension culture because it causes cell damage and differentiation. To achieve agitation with a lower shear stress, researchers and industries have developed a variety of orbital shaking vessel systems2,10.
However, conventional culture vessels are not designed for orbital shaking cultures. In orbital shaking vessels, cells gravitate towards the center-bottom of the vessels by a type of medium flow known as the &#34;Einstein&#39;s tea leaf paradox&#34;11, which causes the inhomogeneous aggregation of cells. The circular flow, caused by a centrifugal force and a friction between the culture medium and a vessel, sweeps cells into the center11,12. In addition, conventional culture bags for mass culture are square-shaped, which is not suitable for orbital shaking.
In this study, we developed a novel culture bag suitable for orbital shaking cultures. The novelty of this bag is its O-shape which does not have a center region, so cells are prevented from gathering at the bottom center region. We have also demonstrated the handling of these vessels with an HEK293 culture to demonstrate the possibility of these bags for biotechnological applications.

<table><tbody><tr><td>HEK293</td><td>RIKEN Bio resorce centre</td><td>RCB1637</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>GIBCO</td><td>11995040</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified, USDA-approved regions</td><td>GIBCO</td><td>10437-028</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution, 100X</td><td>GIBCO</td><td>11140050</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>GIBCO</td><td>25200056</td><td></td></tr><tr><td>Dulbecco&#039;s PBS (&amp;ndash;)</td><td>Cell Science &amp;amp; Technology Institute</td><td>1102P05</td><td></td></tr><tr><td>Cell Strainer 40&amp;micro;m</td><td>CORNING</td><td>352340</td><td></td></tr><tr><td>50 mL Syringe</td><td>TERUMO</td><td>SS-50ESZ</td><td></td></tr><tr><td>Shaker</td><td>AS ONE</td><td>2-1987-02</td><td></td></tr><tr><td>Centrifuge Tube 50 mL</td><td>AS ONE</td><td>1-3500-02</td><td></td></tr><tr><td>Automated cell counter</td><td>BioRad</td><td>TC20</td><td></td></tr></tbody></table>

an orbital shaking culture of mammalian cells in o-shaped vessels to produce uniform aggregates

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based method is one of the simplest techniques for the mass production of cellular aggregates, but it does not protect the cultures against over-aggregation, which occurs when they gather at the bottom center of the culture vessel. To solve this problem, we developed an O-shaped dish and an O-shaped bag, neither of which contains a central region. Aggregates grown in either O-shaped culture vessel were noticeably more uniform in size than aggregates grown in conventional vessels. Histological analyses showed that aggregates in conventional culture dishes contained necrotic cores most likely caused by a poor oxygen supply. In contrast, aggregates that were grown in the O-shaped bag, even those with similar diameters to aggregates in conventional culture dishes, did not show necrotic cores. These results suggest that the O-shaped bag provides sufficient oxygen to the aggregates due to the oxygen permeability of the bag material. We, therefore, propose that this novel gas-permeable O-shaped culture bag is suitable for the mass production of uniform aggregates that are necessary in various biotechnological fields.

According to our measurements, aggregates grown in the conventional dish had varied diameters after 5 d of orbital shaking. In contrast, aggregates grown in O-shaped vessels for 5 d had much more uniform diameters. The conventional dish culture showed small aggregates (50 - 200 &#181;m), whereas the cultures in the O-shaped vessels did not contain any small aggregates (Figure 2a). According to the image-based size measurement, the conventional dish culture showed two different peaks (Figure 2b1), indicating a wide deviation of aggregate size. This result implied that aggregates in the conventional dish were growing under two different conditions in the same culture, which could result in heterogeneous cell quality. On the other hand, aggregates in O-shaped vessels showed a single peak and less deviation in diameter than those in the conventional dish (Figure 2b2 and 2b3), suggesting that such aggregates may be of more uniform quality.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57922/57922fig2.jpg" /> 
Figure 2: Morphologies and diameters of aggregates grown in various vessels. These panels show (a) brightfield images and (b) histograms of aggregates grown in either (a1 and b1) a conventional dish, (a2 and b2) an O-shaped dish, or (a3 and b3) an O-shaped bag. <a href="https://www.jove.com/files/ftp_upload/57922/57922fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Hematoxylin and eosin staining of aggregate cross-sections showed that aggregates grown in the conventional dish had some denucleated cells and necrotic cores (Figure 3a). However, aggregates grown in O-shaped vessels did not show any necrotic cores (Figure 3b and 3c). In particular, aggregates grown in the O-shaped bag were as large as those in the conventional dish yet did not have any necrotic cores (Figure 3c). These results suggested that the gas-permeable bag supplied enough oxygen to the culture.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57922/57922fig3.jpg" /> 
Figure 3: Cross-sections of aggregates in various vessels. The cross-sections are 12 &#181;m thick and were prepared from frozen samples. The sections were stained with hematoxylin and eosin to visualize the cells. (a) The cell aggregates grown in a conventional suspension culture showed necrotic cores. Cell aggregates grown in either (b) an O-shaped dish or (c) an O-shaped bag showed very little necrosis in their cores. <a href="https://www.jove.com/files/ftp_upload/57922/57922fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Cell counts showed that more than 85% of the cells survived for 5 d of suspension culture in each vessel (Figure 4a). The final cell density was approximately 1.5 - 2.0 x 106 cells/mL. Although there was no significant difference, the growth ratio in the O-shaped bag was higher than those in other vessels (Figure 4b). The specific growth rates of the cells in the conventional dish, the O-shaped dish, and the O-shaped bag between day 2 and day 5 were 0.018, 0.025, and 0.020 h-1, respectively.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57922/57922fig4.jpg" /> 
Figure 4: Cell viability and growth. These panels show the (a) cell viability and (b) cell density of HEK293 cells in various culture vessels after 2 d of culture (day 2) and 5 d of culture (day 5). The values shown represent the mean of the results from 3 independent experiments. The values for each experiment were calculated from trypan blue-stained cells counted with an automatic cell counter. The error bars indicate standard deviation. <a href="https://www.jove.com/files/ftp_upload/57922/57922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Bead distribution in 2 different dish formats in various shaking conditions. This panel shows the bead distribution during various shaking conditions (30, 40, and 45 rpm) in a conventional and O-shaped dish. <a href="https://www.jove.com/files/ftp_upload/57922/Figure_S1.pdf">Please click here to download this file.</a>

Watch this Scientific Journal Video about An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates at JoVE.com

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Here we present a protocol for using O-shaped vessels, specialized for suspension cultures of cellular aggregates, with orbital shaking. The HEK293 cells grown in this bag form more homogeneous aggregates than those grown in conventional culture vessels.

Suspension cultures of mammalian cell aggregates are required for various applications in medical and biotechnological fields. The disposable bag-based ...

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Research

JoVE Journal

Bioengineering

9.3K Views.  University of Tokyo. Deze methode is zeer geavanceerd. Het heeft de divisies van de biotechnologie veld, zoals tissue engineering, gratis generatieve therapie en drug screening. Het belangrijkste voordeel van deze techniek is dat uniforme grootte aggregaten kunnen worden geproduceerd in eenvoudige, suspensie cultuur.Dit bord vertegenwoordigt de mogelijkheid van de nieuwe O-vormige vaten voor de productie van uniforme aggregaten van zoogdiercellen in orbitale schudden suspensie cultuur. In orbitale schudden...

Video: An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/&#946;-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

Generation of Aggregates of Mouse Embryonic Stem Cells that Show Symmetry Breaking, Polarization and Emergent Collective Behaviour In Vitro

We have developed a protocol to generate aggregates of mouse embryonic stem cells that display self-organization, symmetry breaking and elongation paralleling axial development. This technique allows the study of axial developmental processes and the generation of cell types that are otherwise difficult to perform in monolayer culture.

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation ...

Developmental Biology

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

Environment

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Neuroscience

Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) on Earth. These options vary in their methods, principles, and suitability for use with suspension cell culture. Here, a cell culture method is described for subjecting lymphocytes to simulated microgravity using a commercially available rotary cell culture system, also known as a 2D clinostat or a rotating wall vessel (RWV) device. This cell culture method utilizes the principle of time-averaged gravity vector nullification to simulate microgravity by rotating the cells on a horizontal axis. The cells cultured in this system can be harvested and utilized in many different experimental assays to assess the effects of simulated microgravity on cellular function and physiology. The culturing technique may vary slightly depending on the cell type or line that is used, but the method described here may be applied to any suspension-type cell culture.

This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture.

Given the current limitations of conducting biological research in space, a few options exist for subjecting cell culture to simulated microgravity (SMG) ...

Immunology and Infection

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Biology

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.

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

Impact of Cytoskeleton on Nuclear Shape and Condensate Dynamics in Mouse Oocytes

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.

Author Spotlight: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named ...

An Orbital Shaking Culture of Mammalian Cells in O-shaped Vessels to Produce Uniform Aggregates

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