Name: nutrient regulation by continuous feeding for large-scale expansion of mammalian cells in spheroids
Uploaded: 2016-09-25T13:00:00
Description: Watch this Scientific Journal Video about Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids at JoVE.com

The authors thank Michael Garwood and Sam Stein for their helpful comments, and Kristen M. Maynard for assistance with manuscript preparation.

1. Cell Line and Maintenance
<ol>
	<li>Obtain &#946;-TC6 cells (or other desired adherent mammalian cell line). In preparation for the study, culture, passage, and cryopreserve the cells according to provider instructions.</li>
</ol>
2. Assemble the Continuous Feeding System
NOTE: The continuous feeding system design in the method below was based on similar systems described in literature17&#8211;19,21,47&#8211;49. ...

Generating mammalian cell products for the production of biological agents and for cell therapies requires the culture and monitoring of mammalian cells in large scale55&#8211;58. Further, these applications call for defined and validated culture conditions. Simply increasing the volume of cells using research technologies will not meet all of these requirements. Manual medium changes causing fluctuations in nutrients and buildup of waste products reduce cell quality, viability and yield....

In order to generate large numbers of viable and functional human cells for transplantation, regulation of the culture conditions is imperative. Depletion of nutrients, along with buildup of metabolic waste are major contributors to senescence and metabolic changes that reduce the quality of the cell product1&#8211;3. This procedure demonstrates a method to culture mammalian cells in spheroids using a stirred bioreactor combined with an adjusted rate perfusion feeding system to regulate glucose in a physiological range4 throughout the duration of the culture. For the purpose of these studies, the physiological range was defined as between 100 and 200 mg/dl. The same methods can be used to regulate other nutrients and metabolic wastes such as lactate.
Static cultures in small volumes (1 - 30 ml) are typically used in the laboratory setting to maintain and differentiate cell lines for experimental purposes. Cell passaging is performed with complete medium changes as needed at regular intervals. Most &#8220;conventional&#8221; culture medium has a high glucose concentration (450 mg/dl for DMEM used in these studies) to allow for less frequent medium changes without the risk of nutrient limitations. However, this batch-feeding method still requires frequent manipulation, introduces variability in the cell environment, and increases the risk of contamination5&#8211;9. Stirred suspension bioreactors (SSB) provide better mixing and decreased handling3,10&#8211;20, but like static cultures, require manual medium changes that contribute to potentially damaging fluctuations in nutrient and waste product levels. Perfusion feeding of SSB cultures reduces these problems by continuous infusion and removal of medium, but large changes in nutrient levels due to cell growth remain an issue. The use of an adjusted feeding rate from calculations of nutrient usage based on estimated cell requirements can provide the stable cell environment required to optimize cell viability and function21&#8211;24.
There is a large body of literature describing methods for scalable SSB cultures of mammalian cells specifically for culture and expansion of pluripotent cells25&#8211;32, with others focused on islet (beta) cells17,33,34, or production of biological products24,35&#8211;38. Many of these investigated cell types may be grown in spheroid cultures, and specific procedures for the cell type being used should be optimized prior to implementing a continuous feeding system. In this demonstration, a perfusion feeding method was used to expand a beta cell line grown as spheroids in a stirred bioreactor39&#8211;43. The method described herein provides a straightforward implementation of feeding rate adjustments based on off-line glucose measurements to achieve targeted culture conditions. Adjusting the feed rate with this method to maintain a physiological glucose level is shown to increases cell yields. Mammalian cells are dependent on a key nutrient, glucose, for energy production, so the use of this cell line represents a model for many cultured mammalian cells44. In addition, this line exemplifies the further complexity of beta cells, which are sensitive to chronic high levels of glucose45. For this study, &#946;-TC6 cells were allowed to form spheroids in culture to approximate the average size of islets of Langerhans in vivo. The perfusion bioreactor system17&#8211;19,21,46 with a feed rate adjusted to glucose consumption, resulted in maintaining physiological conditions and higher cell yields without changes in viability.

<table border="0" cellpadding="0" cellspacing="0" width="840">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Name of Reagent/ Equipment</td>
			<td style="width:249px;">Company</td>
			<td style="width:153px;">Catalog Number / Link</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BTC-6 Cells</td>
			<td style="width:249px;">ATCC, Manassas, VA</td>
			<td style="width:153px;">CRL-11506</td>
			<td>Mouse Insulinoma cell line (adherent cell type)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DPBS No CA, No Mg</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">14190-144</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144">https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Dulbecco&#39;s Modified Eagles Medium</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;"></td>
			<td>See below for product numbers&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMEM High Glucose (500mM)</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td>11965-092</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/11965092">http://www.lifetechnologies.com/order/catalog/product/11965092</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMEM Low Glucose (100mM)</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td>11885-084</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/11885084%20(note%20that%20this%20medium%20already%20contains%20pyruvate)">http://www.lifetechnologies.com/order/catalog/product/11885084 (note that this medium already contains pyruvate)</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">L-gultamine</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">25030081</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product">http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium Pyruvate</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">11360070</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product">https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Heat Inactivated Porcine Serum</td>
			<td style="width:249px;">Gibco - Life Technologies</td>
			<td style="width:153px;">10082147</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/10082147">http://www.lifetechnologies.com/order/catalog/product/10082147</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trypsin-EDTA</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">25200056</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product">https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">T-150 Tissue Culture Treated Flasks</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">430825</td>
			<td><a href="http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences)&amp;categoryname=">http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences) 
			&#38;categoryname=</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuAire Cell culture incubator</td>
			<td style="width:249px;">Princeton, MN</td>
			<td style="width:153px;"></td>
			<td>US Autoflow , Any water-jacketed CO2 regulating cell culture incubator could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:249px;"></td>
			<td style="width:153px;"></td>
			<td>Sorvall RT 7 (Any similar benchtop centrifuge may be used)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Refrigerator</td>
			<td style="width:249px;"></td>
			<td style="width:153px;"></td>
			<td>Any laboratory refrigerator could be used (a small table-top version was used for these studies)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1L Glass Bottle</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">1395-1L</td>
			<td>Any vendor could be used http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=1395-1L(Lifesciences) 
			&#38;categoryname=</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">2L Glass Bottle</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">1395-2L</td>
			<td>Any vendor could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">250 ml stirred bioreactors&#160;</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">4500-250</td>
			<td><a href="http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences)&amp;categoryname=">http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences) 
			&#38;categoryname=</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stir Plate</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">11-496-104A</td>
			<td>Any incubator safe stir-plate can be used, any vendor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Tissue Culture Dishes 100mm Diameter</td>
			<td style="width:249px;">Nunc, Rochester, NY (Fisher Scientific)</td>
			<td style="width:153px;">1256598&#160;</td>
			<td>Any vendor could be used (ordered through Fisher Sci)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">FALCON 50 ml Conical Tubes</td>
			<td style="width:249px;">Falcon, San Jose, CA</td>
			<td style="width:153px;">1256598</td>
			<td>Any vendor could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Delran Plastic Used for Custom Parts</td>
			<td style="width:249px;">McMaster Carr</td>
			<td style="width:153px;">Various</td>
			<td>Any material of choice could be used, but Deran is chosen because it is autoclave safe, non-reactive, and easy to machine, http://www.mcmaster.com/#acetal-homopolymer-sheets/=rjrcac</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stainless Steel Pipe for custom lids</td>
			<td style="width:249px;">McMaster Carr</td>
			<td style="width:153px;">Various</td>
			<td>Any vendor could be used, http://www.mcmaster.com/#standard-stainless-steel-tubing/=rjrd91</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Custom Modified Delran Bioreactor Lids for Continuous Feeding</td>
			<td style="width:249px;">Custom made&#160;</td>
			<td style="width:153px;"></td>
			<td>Not aware of any vendors producing a similar product</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Custom Modified Glass Bottle Lids for Continuous feeding</td>
			<td style="width:249px;">Custom made&#160;</td>
			<td style="width:153px;"></td>
			<td>Some vendors (eg. Fischer Sci, Corning) make similar products in the links below</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Masterflex Digital Peristaltic Pump</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">EW-77919-25</td>
			<td>Any precision peristaltic pump could be used, http://www.coleparmer.com/Product/L_S_Eight_Channel_Four_Roller_ 
			Cartridge_Pump_System_115_230 
			_VAC/EW-77919-25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PVDF Tubing Connectors (various)</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">see link</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Category/Cole_Parmer_PVDF_Premium 
			_Luer_Fittings/55889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pharmed BPT Tubing L/S 16</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">WU-06508-16</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing 
			_L_S_13_25/WU-06508-16</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pharmed BPT Tubing L/S 14</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">WU-06508-14</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing 
			_L_S_13_25/WU-06508-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pharmed BPT Tubing L/S 13</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">WU-06508-13</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing 
			_L_S_13_25/WU-06508-13</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:271px;">Millipore Millex GP PES membrane 0.22ul sterile syringe filter (used for venting, and medium filtration)</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">SLGP033RS</td>
			<td>Any vendor could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">25ml Graduated Pipette</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">13-678-11</td>
			<td>Any vendor could be used, and various sizes may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pipetter</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">13-681-15E</td>
			<td>Any vendor, or similar product could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Hemocytometer</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">02-671-6</td>
			<td>Any vendor, or similar product could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trypan Blue</td>
			<td style="width:249px;">Gibco - Life Technologies</td>
			<td style="width:153px;">15250-061</td>
			<td>Any vendor, or similar product could be used, https://www.lifetechnologies.com/order/catalog/product/15250061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Inverted Light Microscope</td>
			<td style="width:249px;">Leica</td>
			<td style="width:153px;"></td>
			<td>Any vendor, or similar product could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">One Touch Ultra Blood Glucose Meter</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">22-029-293&#160;</td>
			<td>Any vendor, or similar product could be used (eg. Bayer)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">One Touch Ultra-Strips</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">22-029-292&#160;</td>
			<td>Any vendor, or similar product could be used (eg. Bayer)</td>
		</tr>
	</tbody>
</table>

nutrient regulation by continuous feeding for large-scale expansion of mammalian cells in spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Medium Glucose Levels and Fluctuations Restrict Cell Expansion in Standard SSB Cultures
Glucose levels fluctuate in static cultures and SSB cultures throughout the culture period3. These fluctuations intensify with increasing cell number during the 21-day culture period and were nearly identical in both static and SSB cultures. These observations are presented in our previous publication3. The glucose levels can be super-physiological for the duration of ...

Watch this Scientific Journal Video about Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids at JoVE.com

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

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