Name: scale-up of mammalian cell culture using a new multilayered flask
Uploaded: 2011-12-05T13:00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about Scale-Up of Mammalian Cell Culture using a New Multilayered Flask at JoVE.com

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

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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 ...

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 ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...