1. Protocol to use Multi-Flasks for cell culture
<ol>
	<li>Preparation of vessels and adding cell suspension
	<ol>
		<li>Prepare cell growth medium as needed.</li>
		<li>Line up Multi-Flask vertically on its side with the cap facing up on the work surface (sterile laminar flow hood).
		<ol>
			<li>These flasks are available in 3 and 5-layer formats and use a similar work-flow pattern as a T-175 flask.</li>
		</ol>
		</li>
		<li>Loosen and remove caps. Add required amount of pre-warmed medium into flask using a 50 or 100 mL pipette or by pouring.
		<ol>
			<li>Pipettes that are &le; 10 ml can reach the bottom of the vessel when Multi-Flasks are placed vertically with cap facing upwards. Pipettes &ge; 10ml can reach into the vessel immediately past the logo on the Multi-Flask, thereby providing a convenient portal to add larger volumes of media to vessel.</li>
		</ol>
		</li>
		<li>To avoid bubbling of medium, allow liquid stream to flow along the inner wall of the Multi-Flask lid (logo-side).</li>
		<li>Add cell suspension from a concentrated cell stock into growth medium through the top layer using a 10 mL pipette and cap flasks.
		 Tips: 
		- Transport Multi-Flasks on a cart to incubator site and perform remaining steps. 
		- The cell seeding density will vary depending on the cell type, medium and culture duration need. Begin with the seeding density and media volume to that used in standard T-175 flasks and multiply by 3 or 5 depending on the Multi-Flask format used.</li>
	</ol>
	</li>
	<li>Mixing of cells
	<ol>
		<li>Mix position: Hold the Multi-Flask upright with the logo facing you and turn counter clockwise to a 45&deg;angle with mix port facing you.</li>
		<li>Holding at the same angle, gently tilt Multi-Flask from front to back (neck away from you) until liquid in the top layer drains fully downwards through the mix port. Pivot on mix port-side.</li>
		<li>Likewise, gently rock Multi-Flask from back to front (neck towards you) until medium drains fully from the bottom layer towards the top through the mix port. Repeat Steps 1.2.2 and 1.2.3 one more time to ensure proper mixing.</li>
		<li>Bring Multi-Flask back to mix position (Step 1.2.1) and proceed to Step 1.3</li>
		<li>Alternatively, cell suspension can be prepared externally from the Multi-Flask and cell suspension can be added to the vessel using a pipette or by gentle pouring.
		<ol>
			<li>Mix port allows in-vessel mixing of cells with media and eliminates the need to make large volumes of cell suspensions externally.</li>
			<li>It also allows media equalization across the layers of the Multi-Flask</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Equilibration of fluid
	<ol>
		<li>After mixing/adding of cell suspension, place Multi-Flask vertically on a flat work surface to equalize liquid volume equally in all the layers.</li>
	</ol>
	</li>
	<li>Partition liquid into each layer
	<ol>
		<li>Hold the Multi-Flask with the logo facing you and turn clockwise to a 45&deg;angle to partition the liquid into each of the layers. 
		Tip: 
		- For best results, it is important to use a flat work surface for Steps 1.3-1.4</li>
	</ol>
	</li>
	<li>Transportation
	<ol>
		<li>Once media containing cell suspension is partitioned, transport Multi- Flask at the same 45&deg; angle (clockwise) as in Step 1.4.1 with media away from the mix port into incubator. This position is also suitable when removing flasks from incubator for viewing on a microscope.</li>
	</ol>
	</li>
	<li>Placing Multi-Flask onto incubator shelf
	<ol>
		<li>Holding Multi-Flask at the 45&deg; angle (clockwise, away from mix-port), gently rotate it down horizontally onto the incubator surface (using the corner away from the mix port as a pivot). Lay Multi-Flask with Logo facing up.
		<ol>
			<li>Falcon Multi-Flask design allows vessels to be stacked and holds them in place by a sturdy stacking rib.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Distribution of cells and reagents
	<ol>
		<li>After placing Multi-Flask flat on work surface, gently rock back and forth and side-to side to distribute cells evenly onto culture surfaces taking care not to spill liquid from each layer. Stack flasks.
		<ol>
			<li>This Flask is made of optically clear material and cells on the last layer can be easily viewed on a microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Media exchange
	<ol>
		<li>Aspirate while tilting the Multi-Flask to the left (mix port-side) with the logo facing you.</li>
		<li>Then tilt, the Multi-Flask to the right, continuing to aspirate all residual media.</li>
		<li>Add appropriate amount of media and follow Steps 1.3-1.7</li>
	</ol>
	</li>
</ol>
2. Harvesting cells from Multi-Flasks
<ol>
	<li>To dissociate cells, line up Multi-Flasks vertically and bring each one to Mix position (Step 1.2.1). Gently invert flasks with neck facing you so as to allow media to drain to the top layer of Multi-Flask.</li>
	<li>Insert a 5 or 10 mL aspirating tip through the neck until you reach liquid level near the mix port. Aspirate exhausted medium.</li>
	<li>Add dissociation reagent/wash buffer and bring to Mix position as in Step 1.2.1, then proceed and follow steps 1.3-1.7. Neutralize with growth medium /neutralizing agent and pour cell suspension into a receiving tube OR invert flask such that cells drain to the top layer of the vessel and collect cells using a 10 mL pipette. 
	Tip: To enhance recovery of cells or reagents, bring Multi-Flask to mix position (Step 1.2.1), invert with neck towards operator to allow complete drainage of media from all layers to the top layer. Then, tilt Multi-Flask clockwise to 45&deg; angle (away from the mix port) while the Multi-Flask remains inverted. Use a pipette (1-10 mL) to collect any remaining reagents.</li>
</ol>
3. Recommended working volume in Falcon Multi-Flasks
Growth media
3-Layer: 75-150 mL per Multi-Flask 5-Layer: 125-250 mL per Multi-Flask
Dissociation agent
3-Layer: &ge;15 mL per Multi-Flask 5-Layer: &ge;25 mL per Multi-Flask
Tip: Begin with medium volume used in standard T-175 flasks and multiply by 3 or 5 times depending on the Multi-Flask format evaluated so that mL per unit surface area remains the same.
4. Representative Results:
1. Design of Falcon Multi-Flask
<img alt="Figure 1" src="/files/ftp_upload/3418/3418fig1.jpg" /> Figure 1.: Multi-Flask Cell Culture vessels are available in a 3- and 5-layer stackable format for scale-up of cells providing 525 and 875 cm2 growth surface area, respectively. Pipette access facilitates addition and removal of cells and reagents into-and out of the vessel. Presence of mix-port allows for rapid in-vessel mixing and equalization of media across all layers of the Multi-Flask.
2. Cell Yield using Multi-Flask:
These vessels are available in 3-layer and 5-layer formats which correspond to 3 and 5-times the surface area of T-175 flasks. Accordingly, &ge; 3 and 5 times (130&plusmn; 6.8 x 106 and 218 &plusmn; 23.6 x 106 cells, respectively) the number of BHK-21 (baby hamster kidney) cells were grown and recovered from Multi-Flask compared to T-175 flasks (43.2 &plusmn; 3.5 x 106 cells; Fig.2A). Cell yield per unit surface area was equivalent in 3- and 5-layer Multi-Flasks and T-175 flasks for BHK-21, LnCAP (human prostate adenocarcinoma cell line), Hep-G2 (human hepatocarcinoma cell line), EcoPack 2-293 (human kidney cell line) cultured for a period of 48-96h (Fig.2B) in growth media (35 ml per layer) as recommended by cell vendor (ATCC, Sigma and/or Clontech). Cells were enumerated on an automated Vi-CELL counter (1).
 
<img alt="Figure 2A" src="/files/ftp_upload/3418/3418fig2A.jpg" /> Figure 2A : Three and five times the number of BHK-21 cells were grown and recovered from 3- and 5-layer Multi-Flasks compared to T-175 flasks. Expected yield was determined using mean cell yield from control T-175 flasks multiplied by three and five times for the 3- and 5-layer Multi-Flasks respectively (n=4 flasks/format).
<img alt="Figure 2B" src="/files/ftp_upload/3418/3418fig2B.jpg" /> Figure 2B : Cell yield per cm2 was equivalent in 3- and 5-layer Multi-Flasks and T-175 flasks for BHK-21, LnCAP, HepG2 and EcoPack2-293 cells. Each bar represents mean of 4 to 6 flasks. BHK-21 cells (11,000 cells/cm2) were cultured for 72 hours, LnCAP cells (20,000 cells/cm2) and EcoPack2-293 cells (&tilde;35,000 cells/cm2) were cultured for 96 hours and HepG2 cells (25,000 cells/cm2) were cultured for 48 hours prior to harvest.
3. Media distribution among layers of Multi-Flask
Cell culture medium (DMEM; Invitrogen) was added to 5-layer Multi-Flasks (250 mL/5-layer vessel) and partitioned into layers according to protocol described above. Media distribution was measured by drilling holes in each layer and media pumped out from individual layers. Weight of fluid recovered from each layer was found to be relatively uniform from layer to layer as shown in Fig 3. They are as follows: 51.8 &plusmn; 0.73, 50.3 &plusmn; 0.58, 50.21 &plusmn; 0.13, 49.88 &plusmn; 0.35, 49.45 &plusmn; 0.37 (gm).
<img alt="Figure 3" src="/files/ftp_upload/3418/3418fig3.jpg" /> Figure 3. Uniform media distribution in each of the five layers of a 5-layer Multi-Flask. Cell culture medium was added to Multi-Flasks (250 ml/5-layer vessel), equilibrated &amp; partitioned to individual layers. The media was pumped out through holes drilled into individual layers and fluid weight was recorded from each layer (n=6 flasks).
4. Cell distribution between layers of Multi-Flask
Cells can be added and mixed within the Multi-Flask. We simulated distribution of cells between layers of Multi-Flask using beads (10&mu;m; PolySciences Inc.) similar in size to cells. Bead suspension was added into Multi-Flask vessel using a 10 mL pipette through the top layer and mixed with media in the vessel as described in the protocol above. Bead distribution was measured by drilling holes in each layer and fluid containing bead suspension was pumped out from individual layers. Bead concentration recovered from each layer was read on a Coulter Counter and recorded. Shown below are equivalent inter-layer bead distributions in 3-layer Multi-Flasks (Fig.4A). The mix-port enables homogenous distribution of cells and reagents between Multi-Flask layers.
<img alt="Figure 4A" src="/files/ftp_upload/3418/3418fig4A.jpg" /> Figure 4A : Bead distribution in each of the three layers of a 3-layer Multi-Flask. A suspension of beads (3.6 x106/ml) was added to medium dispensed into Multi-Flasks (bead suspension:media volume is 1:10, vol:vol) and mixed, followed by equilibration and partition steps using the protocol described. Bead concentration recovered from each layer (medium pumped through holes drilled on each layer) was read on a Coulter Counter and recorded. Shown below are equivalent inter-layer bead distributions in 3- layer Multi-Flasks (n=5 flasks)
Shown below are representative images of Ecopack 2-293 staining patterns of cells grown to &gt;80% confluence on 3-layer Multi-Flasks in supplemented growth media. Cell monolayers were fixed and stained with crystal violet and Multi-Flask layers were then cut and images scanned (2). Note, cell patterning was homogenous on all layers of Multi-Flask (Fig.4B). Similar results were obtained with multiple cell types evaluated (data not shown).
<img alt="Figure 4B" src="/files/ftp_upload/3418/3418fig4B.jpg" /> Figure 4B : This figure illustrates homogeneous cell growth between layers of Multi-Flasks. Ecopack-2-293 cells grown to &gt;80% confluence in 3-layer Multi-Flasks and T-175 were fixed and stained with crystal violet. Multi-Flask vessels were cut and each stained layer was scanned.
5. Air supply in Multi-Flask:
Analysis of spent media using BioProfile FLEX analyzer (3,4) (Nova Biomedical) from EcoPack2-293 cells cultured for 96 hours revealed no difference in air saturation of cells grown in 5-layer Multi-Flasks vs. T-175 flasks ( 81.03 &plusmn; 1.9 vs. 83.4 &plusmn; 5.8 % ambient O2).
<img alt="Figure 5" src="/files/ftp_upload/3418/3418fig5.jpg" /> Figure 5 : Air saturation (% ambient O2) of spent media were similar in pre-mixed media from 5-layer Multi-Flasks vs. T-175 flasks. EcoPack2-293 cells were seeded at a density of 35,000 cells/cm2 and cultured for 96 hours prior to media analysis (n=3 flasks).

<ol>
	<li>Szabo, S. E., Monroe, S. L., Fiorino, S., Bitzan, J., Loper, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+automated+instrument+for+viability+and+concentration+mesurements+of+cryopreserved+hematopoietic+cells.">Evaluation of an automated instrument for viability and concentration mesurements of cryopreserved hematopoietic cells.</a> Lab. Hematol. 10, 109-109 (2004).</li><li>Bonnekoh, B., Wevers, A., Jugert, F., Merk, H., Mahrle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+growth+assay+for+epidermal+cell+cultures+by+their+crystal+violet+binding+capacity.">Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity.</a> Arch. Dermatol. Res. 281, 487-487 (1989).</li><li>Sengupta, N., Rose, S. T., Morgan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Flux+analysis+of+CHO+cell+metabolism+in+the+late+non-growth+phase.">Metabolic Flux analysis of CHO cell metabolism in the late non-growth phase.</a> Biotechnol. Bioeng. 108, 83-83 (2010).</li><li>Zhu, M. M., Goyal, A., Rank, D. L., Gupta, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+elevated+pCO2+and+osmolality+on+growth+of+cells+and+production+of+antibody&#x2013;fusion+protein+B1:+a+case+study.">Effects of elevated pCO2 and osmolality on growth of cells and production of antibody&#x2013;fusion protein B1: a case study.</a> Biotechnol. Prog. 21, (2005).</li><li>Ryan, J. M., Sharf, B. B., Cristofalo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+culture+medium+volume+on+cell+density+and+life-span+of+human+diploid+fibroblasts.">The influence of culture medium volume on cell density and life-span of human diploid fibroblasts.</a> Exp Cell Res. 91, 389-389 (2004).</li><li>Flaherty, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tips and Techniques for enhancing your cell culture. , (2009).</li><li>Sanyal, S., Abraham, E. J., Slater, K., Cai, A., Lacey, B., Hartshorn, C., Flaherty, P., McHugh, B., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Scaling up cells in BD Falcon Cell Culture Multi-Flasks. , (2011).</li><li>DiPersio, C. M., Jackson, D. A., Zaret, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+coordinately+modulates+liver+transcription+factors+and+hepatocyte+morphology.">The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology.</a> Mol. Cell. Biol. 11, 4405-4405 (1991).</li><li>Grose, R., Hutter, C., Bloch, W., Thorey, I., Watt, F. M., Fassler, R., Brakebusch, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crucial+role+of+&#x03B2;1+integrins+for+keratinocyte+migration+in+vitro+and+during+cutaneous+wound+repair.">A crucial role of &#x03B2;1 integrins for keratinocyte migration in vitro and during cutaneous wound repair.</a> Dev. 129, 2303-2303 (2002).</li></ol>

All authors are employees of Becton Dickinson, Biosciences Segment, Discovery Labware Unit.

The current study demonstrates the increase in productivity that the Multi-Flask design offers researchers. Whilst it is important to follow the steps outlined above for optimal performance when using Multi-Flasks, there are few critical steps in this protocol that are deemed most essential. These include (i) mixing of cells and reagents using the mix port within the vessel (ii) transporting Multi-Flask at a 45&deg; angle clockwise after partitioning fluid into each of the layers (iii) laying Multi-Flask flat post partitioning into the incubator.
Proper use of Multi-Flask leads to production of a homogeneous cell population within each vessel that is cultured in an environment that is congruent to T-175 flasks (5). These vessels provide 3 and 5 times more cells in a similar footprint as the T-175 flask. The 3 and 5-layer vessel provides 525 and 875cm2 of growth area, respectively and offer both space and labor savings to users. Tissue Culture Surface treatment is comparable to standard flasks thus enabling scale-up without the need for re-optimizing existing culture conditions or compromising quality, homogeneity or performance of cells (6, 7). This also provides for comparability with previously collected data. These vessels can be also coated with reagents such as collagen, fibronectin, poly-D-lysine to provide a specialized substratum for attachment, growth and differentiation of certain cell types such as hepatocytes8, kertainocytes9, stem cells grown in serum-free media formulations. Coating solutions can be removed with minimal residual liquid retention so as to reduce wastage of valuable reagents as well as effective removal of coating solution prior to culturing cells. The recommended optimal media volume to culture cells in Multi-flasks range from 0.142-0.287 ml/cm2 which translate to 25-50 ml per layer. Unlike another multilayered flask, this product offers a mix-port in the vessel that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and between layers of each vessel. Diverse cell lines, primary cultures and stem cells have been scaled up efficiently using Multi-Flasks. These vessels are particularly advantageous in applications that demand a large number of cells such as in high throughput-screening, vaccine production, viral vector transfections and cell therapy.
Savings in time, space, labor and reduced waste generation are key winnings of the Multi-Flask versus conventional cultures in single-layered vessels. We can culture approximately three times the number of cells harvested from 5, T-175 flasks in the same space using 3, 5-layer Multi-Flasks. This advantage in space saving is not limited only to T-175 but can also be extended to other vessels: a standard roller bottle apparatus that fits into common laboratory incubators houses &tilde;4 roller bottles (2200 ml) each of which provides 850cm2 surface area. In the same area, &tilde;20, 5-layer Multi-Flasks can be housed thus providing five-times the growth surface to culture cells. Furthermore, with an increase in the "Go-Green" awareness, options to reduce waste generation is highly desirable. In that respect, there is a 38% (5, T-175 flasks weigh &tilde;640g whereas one, 5-layer Multi-Flask weighs &tilde; 400g) decrease in waste generation using Multi-Flasks compared to T-175 flasks and these advantages lead to decrease in waste storage and disposal costs and result in economic savings to the user.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue #</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>3-layer Tissue Culture-treated 525cm2</td>
 <td>BD Biosciences</td>
 <td>353143</td>
 <td rowspan="21"><a href="http://www.bdbiosciences.com/cellculture/multiflasks/index.jsp">BD Biosciences Cell Culture - BD Falcon Multi-Flasks</a></td>
 </tr>
 <tr>
 <td>5-layer Tissue Culture-treated 875cm2</td>
 <td>BD Biosciences</td>
 <td>353144</td>
 </tr>
 <tr>
 <td>100ml pipette</td>
 <td>BD Biosciences</td>
 <td>357600</td>
 </tr>
 <tr>
 <td>10 ml pipette</td>
 <td>BD Biosciences</td>
 <td>357551</td>
 </tr>
 <tr>
 <td>5 ml aspirating pipette</td>
 <td>BD Biosciences</td>
 <td>357501</td>
 </tr>
 <tr>
 <td>50 ml Polypropylene conical tube</td>
 <td>BD Biosciences</td>
 <td>352070</td>
 </tr>
 <tr>
 <td>T-175 flask Tissue Culture-treated</td>
 <td>BD Biosciences</td>
 <td>353028</td>
 </tr>
 <tr>
 <td>Gram Crystal Violet</td>
 <td>BD</td>
 <td>212525</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Invitrogen</td>
 <td>11885</td>
 </tr>
 <tr>
 <td>GMEM</td>
 <td>Sigma</td>
 <td>G5154</td>
 </tr>
 <tr>
 <td>RPMI-1640</td>
 <td>ATCC</td>
 <td>30-2001</td>
 </tr>
 <tr>
 <td>MEM</td>
 <td>ATCC</td>
 <td>30-2003</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA</td>
 <td>Lonza</td>
 <td>CC-5012</td>
 </tr>
 <tr>
 <td>Fetal Bovine serum</td>
 <td>Invitrogen</td>
 <td>16000-044</td>
 </tr>
 <tr>
 <td>Tryptose Phosphate Broth</td>
 <td>Sigma</td>
 <td>T8159</td>
 </tr>
 <tr>
 <td>BHK-21 cells</td>
 <td>Sigma</td>
 <td>85011433</td>
 </tr>
 <tr>
 <td>Hep-G2, LnCAP</td>
 <td>ATCC</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ecopack 2-293</td>
 <td>Clontech</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Electron Microscopy Sciences</td>
 <td>15710</td>
 </tr>
 <tr>
 <td>Polystyrene Bead </td>
 <td>PolySciences Inc.</td>
 <td>24628-20</td>
 </tr>
 <tr>
 <td>Bio-Profile Flex Analyzer</td>
 <td>Nova BioMedical</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

scale-up of mammalian cell culture using a new multilayered flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Watch this Scientific Journal Video about Scale-Up of Mammalian Cell Culture using a New Multilayered Flask at JoVE.com

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

scale-up-of-mammalian-cell-culture-using-a-new-multilayered-flask

Nanyang Technological University

Research

JoVE Journal

Biology

37.5K Views.  BD Biosciences. Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

Video: Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Development of New Therapeutic Applications Using Microfluidics

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and accuracy. Cell counts are important for monitoring cell health and proliferation rate, assessing immortalization or transformation, seeding cells for subsequent experiments, transfection or infection, and preparing for cell-based assays. It is important that cell counts be accurate, consistent, and fast, particularly for quantitative measurements of cellular responses.
Despite this need for speed and accuracy in cell counting, 71% of 400 researchers surveyed1 who count cells using a hemocytometer. While hemocytometry is inexpensive, it is laborious and subject to user bias and misuse, which results in inaccurate counts. Hemocytometers are made of special optical glass on which cell suspensions are loaded in specified volumes and counted under a microscope. Sources of errors in hemocytometry include: uneven cell distribution in the sample, too many or too few cells in the sample, subjective decisions as to whether a given cell falls within the defined counting area, contamination of the hemocytometer, user-to-user variation, and variation of hemocytometer filling rate2.
To alleviate the tedium associated with manual counting, 29% of researchers count cells using automated cell counting devices; these include vision-based counters, systems that detect cells using the Coulter principle, or flow cytometry1. For most researchers, the main barrier to using an automated system is the price associated with these large benchtop instruments1.
The Scepter cell counter is an automated handheld device that offers the automation and accuracy of Coulter counting at a relatively low cost. The system employs the Coulter principle of impedance-based particle detection3 in a miniaturized format using a combination of analog and digital hardware for sensing, signal processing, data storage, and graphical display. The disposable tip is engineered with a microfabricated, cell- sensing zone that enables discrimination by cell size and cell volume at sub-micron and sub-picoliter resolution. Enhanced with precision liquid-handling channels and electronics, the Scepter cell counter reports cell population statistics graphically displayed as a histogram.

The Scepter Cell Counter is a handheld automated device that can be used to count cells, monitor cell diameter and volume, and be used to check the health and quality of cellular populations from one culture to the next.

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...