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 β-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.
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 product1–3. 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 “conventional” 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 contamination5–9. Stirred suspension bioreactors (SSB) provide better mixing and decreased handling3,10–20, 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 function21–24.
There is a large body of literature describing methods for scalable SSB cultures of mammalian cells specifically for culture and expansion of pluripotent cells25–32, with others focused on islet (beta) cells17,33,34, or production of biological products24,35–38. 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 bioreactor39–43. 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, β-TC6 cells were allowed to form spheroids in culture to approximate the average size of islets of Langerhans in vivo. The perfusion bioreactor system17–19,21,46 with a feed rate adjusted to glucose consumption, resulted in maintaining physiological conditions and higher cell yields without changes in viability.
1. Cell Line and Maintenance
2. Assemble the Continuous Feeding System
NOTE: The continuous feeding system design in the method below was based on similar systems described in literature17–19,21,47–49. Assembly of the system used here is described in detail in a previous publication3.
3. Autoclave All Materials
4. Spheroid Formation
NOTE: This technique is similar to those described in the literature17–19,21,50–52 for other mammalian cell cultures. All procedures following sterilization should be done in a laminar flow hood and using sterile gloves to maintain sterile conditions for cell culture.
5. Continuous Feeding Culture and Adjusted Feed Rate
6. Cell Counts, Viability, and Glucose Concentration Measurements
7. Spheroid Settling Rate Measurements
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 “drop” of added medium time to equilibrate temperature with the surrounding culture medium that was at 37 °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 µm 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 ± 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 < 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 ± 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).
(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.
(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.
(3)
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
Figure 2: Glucose Measurements for SSB, CF-SSB, and Adjusted CF-SSB Cultures. (A) Medium glucose measurements from β-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 ≤ 4% for all measurements). The data from the figures is presented in previous publication with permission.3
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
Generating mammalian cell products for the production of biological agents and for cell therapies requires the culture and monitoring of mammalian cells in large scale55–58. 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 processes61–63 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.
The authors have nothing to disclose.
The authors thank Michael Garwood and Sam Stein for their helpful comments, and Kristen M. Maynard for assistance with manuscript preparation.
Name of Reagent/ Equipment | Company | Catalog Number / Link | Comments/Description |
BTC-6 Cells | ATCC, Manassas, VA | CRL-11506 | Mouse Insulinoma cell line (adherent cell type) |
DPBS No CA, No Mg | Invitrogen, Carlsbad, CA | 14190-144 | https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144 |
Dulbecco's Modified Eagles Medium | Invitrogen, Carlsbad, CA | See below for product numbers | |
DMEM High Glucose (500mM) | Invitrogen, Carlsbad, CA | 11965-092 | http://www.lifetechnologies.com/order/catalog/product/11965092 |
DMEM Low Glucose (100mM) | Invitrogen, Carlsbad, CA | 11885-084 | http://www.lifetechnologies.com/order/catalog/product/11885084 (note that this medium already contains pyruvate) |
L-gultamine | Invitrogen, Carlsbad, CA | 25030081 | http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product |
Sodium Pyruvate | Invitrogen, Carlsbad, CA | 11360070 | https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product |
Heat Inactivated Porcine Serum | Gibco – Life Technologies | 10082147 | http://www.lifetechnologies.com/order/catalog/product/10082147 |
Trypsin-EDTA | Invitrogen, Carlsbad, CA | 25200056 | https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product |
T-150 Tissue Culture Treated Flasks | Corning, Corning, NY | 430825 | http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences) &categoryname= |
NuAire Cell culture incubator | Princeton, MN | US Autoflow , Any water-jacketed CO2 regulating cell culture incubator could be used | |
Centrifuge | Sorvall RT 7 (Any similar benchtop centrifuge may be used) | ||
Refrigerator | Any laboratory refrigerator could be used (a small table-top version was used for these studies) | ||
1L Glass Bottle | Corning, Corning, NY | 1395-1L | Any vendor could be used http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=1395-1L(Lifesciences) &categoryname= |
2L Glass Bottle | Corning, Corning, NY | 1395-2L | Any vendor could be used |
250 ml stirred bioreactors | Corning, Corning, NY | 4500-250 | http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences) &categoryname= |
Stir Plate | Fisher Scientific | 11-496-104A | Any incubator safe stir-plate can be used, any vendor |
Tissue Culture Dishes 100mm Diameter | Nunc, Rochester, NY (Fisher Scientific) | 1256598 | Any vendor could be used (ordered through Fisher Sci) |
FALCON 50 ml Conical Tubes | Falcon, San Jose, CA | 1256598 | Any vendor could be used |
Delran Plastic Used for Custom Parts | McMaster Carr | Various | 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 |
Stainless Steel Pipe for custom lids | McMaster Carr | Various | Any vendor could be used, http://www.mcmaster.com/#standard-stainless-steel-tubing/=rjrd91 |
Custom Modified Delran Bioreactor Lids for Continuous Feeding | Custom made | Not aware of any vendors producing a similar product | |
Custom Modified Glass Bottle Lids for Continuous feeding | Custom made | Some vendors (eg. Fischer Sci, Corning) make similar products in the links below | |
Masterflex Digital Peristaltic Pump | Cole Parmer, Vernon Hills, IL | EW-77919-25 | 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 |
PVDF Tubing Connectors (various) | Cole Parmer, Vernon Hills, IL | see link | Any vendor could be used, http://www.coleparmer.com/Category/Cole_Parmer_PVDF_Premium _Luer_Fittings/55889 |
Pharmed BPT Tubing L/S 16 | Cole Parmer, Vernon Hills, IL | WU-06508-16 | Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing _L_S_13_25/WU-06508-16 |
Pharmed BPT Tubing L/S 14 | Cole Parmer, Vernon Hills, IL | WU-06508-14 | Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing _L_S_13_25/WU-06508-14 |
Pharmed BPT Tubing L/S 13 | Cole Parmer, Vernon Hills, IL | WU-06508-13 | Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing _L_S_13_25/WU-06508-13 |
Millipore Millex GP PES membrane 0.22ul sterile syringe filter (used for venting, and medium filtration) | Fisher Scientific | SLGP033RS | Any vendor could be used |
25ml Graduated Pipette | Fisher Scientific | 13-678-11 | Any vendor could be used, and various sizes may be used |
Pipetter | Fisher Scientific | 13-681-15E | Any vendor, or similar product could be used |
Hemocytometer | Fisher Scientific | 02-671-6 | Any vendor, or similar product could be used |
Trypan Blue | Gibco – Life Technologies | 15250-061 | Any vendor, or similar product could be used, https://www.lifetechnologies.com/order/catalog/product/15250061 |
Inverted Light Microscope | Leica | Any vendor, or similar product could be used | |
One Touch Ultra Blood Glucose Meter | Fisher Scientific | 22-029-293 | Any vendor, or similar product could be used (eg. Bayer) |
One Touch Ultra-Strips | Fisher Scientific | 22-029-292 | Any vendor, or similar product could be used (eg. Bayer) |