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 border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">HEK293</td>
			<td style="width:123px;">RIKEN Bio resorce centre</td>
			<td style="width:156px;">RCB1637</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">DMEM, high glucose, pyruvate</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:156px;">11995040</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Fetal Bovine Serum, qualified, USDA-approved regions</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:156px;">10437-028</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">MEM Non-Essential Amino Acids Solution, 100X</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:156px;">11140050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:156px;">25200056</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">Dulbecco&#39;s PBS (&#8211;)</td>
			<td style="width:123px;">Cell Science &#38; Technology Institute</td>
			<td style="width:156px;">1102P05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Cell Strainer 40&#181;m</td>
			<td style="width:123px;">CORNING</td>
			<td style="width:156px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">50 mL Syringe</td>
			<td style="width:123px;">TERUMO</td>
			<td style="width:156px;">SS-50ESZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Shaker</td>
			<td style="width:123px;">AS ONE</td>
			<td style="width:156px;">2-1987-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Centrifuge Tube 50 mL</td>
			<td style="width:123px;">AS ONE</td>
			<td style="width:156px;">1-3500-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Automated cell counter</td>
			<td style="width:123px;">BioRad</td>
			<td style="width:156px;">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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9.3K Views.  University of Tokyo. 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.

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

An Experimental System to Study Mechanotransduction in Fetal Lung Cells

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.

Mechanical forces play a key role in lung development and lung injury. Here, we describe a method to isolate rodent fetal lung type II epithelial cells and fibroblasts and to expose them to mechanical stimulation using an in vitro system.

Mechanical forces generated in utero by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

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.