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

The authors have nothing to disclose.

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. The use of bioreactors is well established for the commercial culture of microorganisms for bio-pharmaceutical applications, and similar strategies have been applied to mammalian cell cultures. The complex interaction of mammalian cells with their environment necessitates specific modifications to regulate nutrient levels in order to optimize cell expansion potential.
To address these issues, we developed a straightforward method to maintain glucose levels in a specific targeted range, approximating physiological levels in a stirred suspension bioreactor with an adjustable rate perfusion feeding mechanism. To accomplish this, spheroids of a beta cell line were generated in a stirred suspension bioreactor. A perfusion feeding system was employed to provide a continuous feeding mechanism to replace standard batch feeding procedures. Cell growth rates were observed, and used to predict approximate glucose use rates. Actual glucose levels also were measured over the time in culture to adjust feed rates in response to actual nutrients consumed and metabolic products deposited in the medium by the cells. This method prevented the fluctuations in glucose levels seen with other static and SSB systems3 where glucose levels fluctuated dramatically with medium changes every 3 days. Using the batch-feeding strategies, glucose concentrations dropped as much as 275 mg/dl over three days, at late stages in the 21-day expansion. These fluctuations in glucose levels limited cell yield.
Calculation of the medium replacement rate for demonstration of the adjusted feeding system incorporated an established growth rate and glucose consumption rate for the cell line, as well as the observed (measured) culture densities and glucose levels at each feeding time-point. For different cell types, or for more complex tissue culture systems, these assumptions may not hold true. To ensure more accurate glucose control, the algorithm also included a feedback control system to account for the variability of individual cultures. Limitations of a perfusion method based on growth rate alone, including the impact of heterogeneity in glucose utilization for cells throughout the spheroids and changes in glucose consumption based on parameters such as differentiating stem cells, can be mitigated by a feedback control system. Depending on the application, cells that exhibit exponential growth rates or more complex growth profiles could be implemented to improve the algorithm performance. The assumptions and values used for the feed rate calculations can be altered to adhere to actual culture conditions.
The presented methods are intended to produce cells for a therapeutic application in which the expansion and culture of spheroids would be for a finite duration, and the cells themselves are the product.. It may be desirable for some investigators to continuously expand or culture cells using a similar method, but this would present other challenges not encountered for these studies. If cultured for extended periods, the spheroids would continue to grow in size, and the viability of some cells in the nucleus of the spheroid may suffer due to increasing nutrient and oxygen diffusion limitations. These cultures may require further manipulations to dissociate large spheroids to prevent this limitation when long-term cultures are desired. No decrease in viability was observed for spheroids generated with this protocol, suggesting that this limit was not reached during the 21-day culture period of this study.
For cellular therapies, these techniques could be used in combination with additional regulatory systems to improve cell yield, function and viability. For example, the SSB method could be combined with facilitating technologies including micro-carrier surface cultures for adherent cells to improve cell growth rates, or encapsulation of cells or spheroids. Further, more complex adjustment algorithms could be implemented to provide tighter culture control. Feeding adjustment could be combined with the control of multiple other parameters, such as pH, dissolved oxygen concentration, temperature, and injection of reagents to make further improvements59,60. To improve outcomes in other applications, this method could be automated15,24, and feed rates can be calculated more or less often, further refining culture conditions.
Manufacturing processes61&#8211;63 must be defined and carefully controlled to make reproducible biological products. The use of continuous medium replacement can be used to mitigate fluctuations in critical nutrients observed in large scale cell production, and incorporating an adjustable feeding system can implement even more control on the cell environment, to maintain desired nutrient levels. The described method could be used with other mammalian cells for both cellular and pharmaceutical products.

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><tbody><tr><td>&amp;#946; TC-6 Cells</td><td>ATCC, Manassas, VA</td><td>CRL-11506</td><td>Mouse Insulinoma cell line (adherent cell type)</td></tr><tr><td>DPBS No Ca, No Mg</td><td>Invitrogen, Carlsbad, CA</td><td>14190-144</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144&quot;&gt;https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144&lt;/a&gt;</td></tr><tr><td>Dulbecco&#039;s Modified Eagles Medium</td><td>Invitrogen, Carlsbad, CA</td><td></td><td>See below for product numbers&amp;nbsp;</td></tr><tr><td>DMEM High Glucose (500 mM)</td><td>Invitrogen, Carlsbad, CA</td><td>11965-092</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/11965092&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/11965092&lt;/a&gt;</td></tr><tr><td>DMEM Low Glucose (100 mM)</td><td>Invitrogen, Carlsbad, CA</td><td>11885-084</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/11885084%20(note%20that%20this%20medium%20already%20contains%20pyruvate)&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/11885084 (note that this medium already contains pyruvate)&lt;/a&gt;</td></tr><tr><td>L-glutamine</td><td>Invitrogen, Carlsbad, CA</td><td>25030081</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product&lt;/a&gt;</td></tr><tr><td>Sodium Pyruvate</td><td>Invitrogen, Carlsbad, CA</td><td>11360070</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product&quot;&gt;https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product&lt;/a&gt;</td></tr><tr><td>Heat Inactivated Porcine Serum</td><td>Gibco - Life Technologies</td><td>10082147</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/10082147&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/10082147&lt;/a&gt;</td></tr><tr><td>Trypsin-EDTA</td><td>Invitrogen, Carlsbad, CA</td><td>25200056</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product&quot;&gt;https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product&lt;/a&gt;</td></tr><tr><td>T-150 Tissue Culture Treated Flasks</td><td>Corning, Corning, NY</td><td>430825</td><td>http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences)&lt;br/&gt;&amp;amp;categoryname=</td></tr><tr><td>NuAire Cell culture incubator</td><td>Princeton, MN</td><td></td><td>US Autoflow. Any water-jacketed CO&lt;sub&gt;2&lt;/sub&gt; regulating cell culture incubator could be used.</td></tr><tr><td>Centrifuge</td><td></td><td></td><td>Sorvall RT 7 (Any similar benchtop centrifuge may be used)</td></tr><tr><td>Refrigerator</td><td></td><td></td><td>Any laboratory refrigerator could be used (a small table-top version was used for these studies)</td></tr><tr><td>1 L Glass Bottle</td><td>Corning, Corning, NY</td><td>1395-1L</td><td>Any vendor could be used. http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=1395-1L(Lifesciences)&lt;br/&gt; &amp;amp;categoryname=</td></tr><tr><td>2 L Glass Bottle</td><td>Corning, Corning, NY</td><td>1395-2L</td><td>Any vendor could be used</td></tr><tr><td>250 ml stirred bioreactors&amp;nbsp;</td><td>Corning, Corning, NY</td><td>4500-250</td><td>http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences)&lt;br/&gt;&amp;amp;categoryname=</td></tr><tr><td>Stir Plate</td><td>Fisher Scientific</td><td>11-496-104A</td><td>Any incubator safe stir-plate can be used, any vendor</td></tr><tr><td>Tissue Culture Dishes 100 mm Diameter</td><td>Nunc, Rochester, NY (Fisher Scientific)</td><td>1256598&amp;nbsp;</td><td>Any vendor could be used (ordered through Fisher Sci)</td></tr><tr><td>FALCON 50 ml Conical Tubes</td><td>Falcon, San Jose, CA</td><td>1256598</td><td>Any vendor could be used</td></tr><tr><td>Delran Plastic Used for Custom Parts</td><td>McMaster Carr</td><td>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><td>Stainless Steel Pipe for custom lids</td><td>McMaster Carr</td><td>Various</td><td>Any vendor could be used. http://www.mcmaster.com/#standard-stainless-steel-tubing/=rjrd91</td></tr><tr><td>Custom Modified Delran Bioreactor Lids for Continuous Feeding</td><td>Custom made</td><td></td><td>Not aware of any vendors producing a similar product</td></tr><tr><td>Custom Modified Glass Bottle Lids for Continuous feeding</td><td>Custom made</td><td></td><td>Some vendors (&lt;em&gt;e.g.&lt;/em&gt;, Fischer Sci, Corning) make similar products in the links below.</td></tr><tr><td>Masterflex Digital Peristaltic Pump</td><td>Cole Parmer, Vernon Hills, IL</td><td>EW-77919-25</td><td>Any precision peristaltic pump could be used. http://www.coleparmer.com/Product/L_S_Eight_Channel_Four_Roller_&lt;br/&gt; Cartridge_Pump_System_115_230&lt;br/&gt; _VAC/EW-77919-25</td></tr><tr><td>PVDF Tubing Connectors (various)</td><td>Cole Parmer, Vernon Hills, IL</td><td>see link</td><td>Any vendor could be used. http://www.coleparmer.com/Category/Cole_Parmer_PVDF_Premium&lt;br/&gt; _Luer_Fittings/55889</td></tr><tr><td>Pharmed BPT Tubing L/S 16</td><td>Cole Parmer, Vernon Hills, IL</td><td>WU-06508-16</td><td>Any vendor could be used. http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing&lt;br/&gt; _L_S_13_25/WU-06508-16</td></tr><tr><td>Pharmed BPT Tubing L/S 14</td><td>Cole Parmer, Vernon Hills, IL</td><td>WU-06508-14</td><td>Any vendor could be used. http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing&lt;br/&gt; _L_S_13_25/WU-06508-14</td></tr><tr><td>Pharmed BPT Tubing L/S 13</td><td>Cole Parmer, Vernon Hills, IL</td><td>WU-06508-13</td><td>Any vendor could be used. http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing&lt;br/&gt; _L_S_13_25/WU-06508-13</td></tr><tr><td>Millipore Millex GP PES membrane 0.22 &amp;#181;l sterile syringe filter (used for venting, and medium filtration)</td><td>Fisher Scientific</td><td>SLGP033RS</td><td>Any vendor could be used</td></tr><tr><td>25 ml Graduated Pipette</td><td>Fisher Scientific</td><td>13-678-11</td><td>Any vendor could be used, and various sizes may be used</td></tr><tr><td>Pipetter</td><td>Fisher Scientific</td><td>13-681-15E</td><td>Any vendor, or similar product could be used</td></tr><tr><td>Hemocytometer</td><td>Fisher Scientific</td><td>02-671-6</td><td>Any vendor, or similar product could be used</td></tr><tr><td>Trypan Blue</td><td>Gibco - Life Technologies</td><td>15250-061</td><td>Any vendor, or similar product could be used. https://www.lifetechnologies.com/order/catalog/product/15250061</td></tr><tr><td>Inverted Light Microscope</td><td>Leica</td><td></td><td>Any vendor, or similar product could be used</td></tr><tr><td>One Touch Ultra Blood Glucose Meter</td><td>Fisher Scientific</td><td>22-029-293</td><td>Any vendor, or similar product could be used (&lt;i&gt;e.g.&lt;/i&gt;, Bayer)</td></tr><tr><td>One Touch Ultra-Strips</td><td>Fisher Scientific</td><td>22-029-292&amp;nbsp;</td><td>Any vendor, or similar product could be used (&lt;i&gt;e.g.&lt;/i&gt;, 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 the culture period for both methods. Because this chronic exposure may inhibit cell growth54, a continuous feeding system was developed to eliminate glucose fluctuations and improve nutrient control during spheroid culture.
Continuous Feeding System for Spheroid Culture
Continuously adding fresh medium and removing old medium for the duration of the culture period can be accomplished using a simple medium replenishment system. The system described in Method 2 and shown in Figure 1 used a pump and tubing set to continuously replenish medium and a separate outflow tube to continuously remove medium while preventing the removal of spheroids from the culture. The medium inlet was at the opposite side of the reactor to minimize any possibility of interfering with the proper function of the OT, and to allow for thorough mixing. Fresh medium (with high glucose, 450 mg/dl) was maintained at refrigerator temperature to ensure long term stability and continuously added to the culture through a medium inlet with a full medium replacement rate every three days to replenish the nutrients. This system limited the manipulations and intervention required during the culture period by replacing the manual batch medium replacement process with a continuous process3,22,23. The cold medium entered the bioreactor in small volumes over time (0.046 ml/min) relative to the total culture volume (200 ml), giving each &#8220;drop&#8221; of added medium time to equilibrate temperature with the surrounding culture medium that was at 37 &#176;C. This ensured that the added cold medium did not reduce the overall culture temperature being maintained within the incubator. Stirring of the culture medium also increased heat transfer efficiency, and improved temperature uniformity in these cultures. Temperature maintenance could be a concern if very-high feed rates were used with small culture volumes, but these unlikely conditions were not tested for these studies. The culture volume was maintained at a constant level in the continuous feeding system by ensuring that the average medium removal rate was equal to the feeding rate. The system used for these studies actually removed medium at a higher flow rate than the feed rate because the removal tubing section used larger diameter tubing for the pump section. Despite the faster removal rate, the culture volume was maintained by adjusting the level of the outflow tube inside the reactor to the desired culture volume level. Continuously adding fresh medium to the SSB resulted in a small increase in the medium level in the reactor, and when the medium reached the level of the OT, medium was removed from the reactor at a faster rate. The medium was removed through the porous glass OT, leaving the cell spheroids in culture until the medium level fell below the bottom of the OT. This system avoided the complexity of using tenuous flow and volume sensors to control the pump speeds, and is the standard for use with many SSB based culture apparatus47,48. The OT was designed to ensure that spheroids were not removed from the culture through the removal circuit, and the pore size and density in the fritted glass tube were large enough (40% and 40 - 60 &#181;m respectively) to ensure that the linear flow velocity was less than the settling rate of the spheroids used for these culture studies. The linear flow was calculated as described in detail3. The average linear medium velocity through the pores in the OT was calculated to be 0.17 cm/min and this was far slower than the slowest observed spheroid settling rate. The average settling velocity was measured as described in method 7 and was 2.53 &#177; 0.26 cm/min for the spheroids used in these studies. The spheroids were observed to increase in size as the culture progresses, and these larger spheroids settled faster due to their increased mass-drag ratio, which further decreased the possibility of removal through the OT. Some spheroids were trapped in the lower pores on the outside edges of the OT, but this was primarily due to mechanical collision when the outflow tube dips into the stirred medium. This did not prevent proper function of the OT, and did not contribute to a measureable loss of spheroids in the cultures tested. The OT for these studies was cleaned after each study, (with DI water flush), and replaced after use for two culture studies to avoid the risk of decreased function. The OT could be replaced after every study if spheroids are observed to clog the pores during a specific study (this was not observed in the presented work). Due to the cyclic removal of medium using this method, the medium culture volume fluctuated by as much as 6%. The fluctuations were caused by the surface tension of the medium that allowed the OT to remove a bit more medium after the average medium level fell below the bottom of the OT.
Eliminating Nutrient Fluctuations Improved Cell Growth in SSB Cultures
Continuously replacing the medium in SSB cultures using the system described above increased culture yields during the 21-day culture period when compared to the standard SSB cultures. The feed rate was maintained constant throughout the culture period at a rate that replaced the entire culture volume every three days to be comparable to the batch-fed SSB cultures. Eliminating the nutrient fluctuations resulted in an increase in cell yields (Figure 3) despite the high glucose concentration3. Spheroid size was also measured at the end of the culture period by 2D microscopic assessment (standard light microscopy described in literature3), and the spheroids in CF-SSB cultures were significantly larger (p &#60; 0.02, student-t test) that in static or standard SSB cultures as reported in literature3. These observations support the cell-count data suggesting a higher growth rate in CF-SSB cultures while the viability was maintained at the same high level (96.23 &#177; 0.85%) regardless of culture condition. The continuously fed SSB (CF-SSB) culture system eliminated the nutrient fluctuations in spheroid cultures, and improved cell growth rates, but the glucose levels remained super-physiological which may have prevented even further improvement in cell expansion (Figure 2). To further improve upon the CF-SSB culture system, an algorithm was used to adjust the feed rate during the culture period with the goal of improving the control of glucose levels in SSB cultures.
Calculation of Adjusted Feed Rate
Several factors can be considered for the calculation of feed rate to maintain consistent glucose levels. The specific factors of interest can be altered for a given study and for a specific cell line. For the purpose of this demonstration, we considered growth rate and glucose consumption determined from previous cultures, as well as the actual glucose levels at the time of adjustment3. Feed rate adjustments can be made at any interval in time. For this demonstration, samples were collected and the feed rate was adjusted every three days based on the sequential calculations described as follows.
Calculation of Predicted Growth Rate
Equation 1 predicts the cell growth during a certain period of culture, with the predicted cell count (N2) representing the expected total number of cells in culture at some time in the future. This method uses a linear growth rate approximation where the current cell count (N1) is added to the estimated growth rate of the cells in spheroid cultures (Rg) multiplied by the culture period (t2-t1).
<img alt="Equation 1" fo:content-width="1in" src="/files/ftp_upload/52224/52224eq1.jpg" /> (1)
Calculated Baseline Feed Rate
The baseline feed rate (RF) was calculated using equation 2 by estimating the glucose consumed in the culture during the culture period (numerator) and dividing it by the glucose concentration in the feed medium (CF, denominator). The consumed glucose is estimated by multiplying the previously determined glucose consumption rate for the given cell type (R1) by the weighted average of N1 and N2 from equation 1. This consumption rate was then divided by the glucose concentration (CF) in the feed medium to calculate the average medium feed rate needed to replace the glucose consumed during the same culture period.
<img alt="Equation 2" fo:content-width="1in" src="/files/ftp_upload/52224/52224eq2.jpg" /> (2)
Adjusting Feed Rate with Observed Glucose Levels
Further adjustments were made to the baseline feed rate to accommodate the measured glucose levels (C1) at the chosen time point using equation 3. This glucose-adjusted feed rate (GAFR) can be used to maintain levels when unexpected changes in growth or death occur in the culture. This equation incorporated a control constant (X), and a value of 0.5 was used for these data3. C1 represents the glucose concentration measured in the culture medium on the sample day, and CD represents the target medium glucose concentration. The final value adjusts the predicted feed rate from the second calculation.
<img alt="Equation 3" fo:content-width="1.5in" src="/files/ftp_upload/52224/52224eq3.jpg" /> (3)
<img alt="Figure 1" src="/files/ftp_upload/52224/52224fig1.jpg" /> 
Figure 1: Continuous Feeding System Diagram with Outflow Tube. (A) Diagram of the demonstrated system. (B) Fritted glass filter placed on outflow tube to allow medium to be removed from the culture without loss of cells. Figure reproduced with permission.3
<img alt="Figure 2" src="/files/ftp_upload/52224/52224fig2.jpg" /> 
Figure 2: Glucose Measurements for SSB, CF-SSB, and Adjusted CF-SSB Cultures. (A) Medium glucose measurements from &#946;-TC6 spheroid cultures using static, SSB, and CF-SSB culture methods and feeding with standard high glucose medium. The continuous feeding is able to eliminate the glucose fluctuations, but the average glucose levels in the medium change dramatically during the culture period, and far above the physiological range (indicated by the grey bar). The adjusted feeding is able to eliminate the fluctuations as well as maintain the glucose concentrations near physiological levels for the duration of the culture period. Error bars for glucose measurements are too small to be visible on the scale shown (Standard Error &#8804; 4% for all measurements). The data from the figures is presented in previous publication with permission.3
<img alt="Figure 3" src="/files/ftp_upload/52224/52224fig3.jpg" /> 
Figure 3: Cell Counts from SSB, CF-SSB, and Adjusted CF-SSB Cultures with Growth Adjusted Feed Rates. Cell growth reported as fold change in cell number during the 21-day culture period comparing SSB with regular medium changes against constant feed rate SSB cultures and adjusted feed rate SSB cultures. Error bars report the standard error of the mean, and p-values reported are for an un-paired two-tailed student-t statistical test. Reproduced with permission.3

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 ...

nutrient-regulation-continuous-feeding-for-large-scale-expansion

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.7K Views.  University of Minnesota. 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.

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

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Cancer Research

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

Biochemistry

Multicellular Spheroid Formation for FLIM-Based Metabolic and Hypoxia Analysis

Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug therapies&#160;and using bio- and nanosensors in a 3D context. Their ease of production, predictable size, growth, and observed nutrient and metabolite gradients are important to recapitulate the 3D niche-like&#160;cell microenvironment. However, spheroid heterogeneity and variability of their production methods can influence overall cell metabolism, viability, and drug response. This makes it difficult to choose the most appropriate&#160;methodology, considering the requirements in size, variability, needs of biofabrication, and use as in vitro 3D tissue models in stem and cancer cell biology. In particular, spheroid production can influence their compatibility with quantitative live microscopies, such as optical metabolic imaging, fluorescence lifetime imaging microscopy (FLIM), monitoring of spheroid&#160;hypoxia with nanosensors, or viability. Here, a number of conventional spheroid formation protocols are presented, highlighting their compatibility with the live widefield, confocal, and two-photon microscopies. The follow-up imaging to analysis&#160;pipeline with multiplexed autofluorescence FLIM and, using various types of cancer and stem cell spheroids, is also presented.

Author Spotlight: Standardizing Spheroid Formation Methods for Metabolic and Oxygenation Analysis Using Fluorescence Lifetime Imaging Microscopy

Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.

Multicellular tumor spheroids are a popular 3D tissue microaggregate&#160;model for reproducing tumor microenvironment, testing and optimizing drug ...

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

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

Biology

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale ...

Spheroid Generation from Cell Lines: A Three-Dimensional (3D) Cell Culture Method

Source: Dillard, P., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/58785/a-spheroid-killing-assay-by-car-t-cells">A Spheroid Killing Assay by CAR T Cells</a>. J. Vis. Exp. (2018).
Spheroids are a type of 3D cell culture model used to more accurately represent a cell&#8217;s environment compared to 2D cell culture monolayer models. In the example protocol, we will see scientists generate spheroids from a colorectal cancer cell line and apply live-cell imaging to track the spheroids&#8217; growth.

Source: Dillard, P., et al. A Spheroid Killing Assay by CAR T Cells. J. Vis. Exp. (2018).
Spheroids are a type of 3D cell culture model used to more ...

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

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