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Method Article

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

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DOI:

10.3791/52224

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September 25th, 2016

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1Radiology, University of Minnesota, 2Medicine, University of Minnesota, 3School of Public Health, University of Minnesota, 4Surgery, University of Arizona

In This Article

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

1. Cell Line and Maintenance

  1. Obtain β-TC6 cells (or other desired adherent mammalian cell line). In preparation for the study, culture, passage, and cryopreserve the cells according to provider instructions.

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.

  1. Collect the components needed for the feeding system which consists of five primary components: a medium reservoir, peristaltic pump, stirred bioreactor, waste reservoir, and custom designed tubing/sampling set.
    1. Establish the medium reservoir using a 1 L glass bottle, the waste reservoir using a 2 L glass bottle, and the stirred bioreactor using a 250 ml volume glass reactor (off the shelf version).
    2. Use a digital peristaltic pump or similar pump with an 8 channel pump head to control medium exchange.
    3. Manufacture the reservoir and modified bioreactor lids from hard and autoclavable plastic with stainless steel pipe pass-through ports to provide ventilation through sterile filters, and allow for medium transfer between reservoirs and bioreactors. Alternatively, purchase specialized lids with flow ports from various vendors.
      NOTE: The bioreactor lid may contain additional pass-through ports for optional instrumentation and monitoring probes (e.g., oxygen monitor).
    4. Attach an outflow tube (OT) fabricated from porous glass aeration tubes with an average pore size range of 40 µm to 60 µm, and a pore density of 40% to the modified SSB lid. Alternatively, choose another outflow tube based on the specific requirements of the culture.
      NOTE: Choose the OT pore size to remove only medium and cell debris, leaving cell aggregates in culture (as described in section 6 and publication3 for more details).
  2. Assemble autoclavable perfusion tubing sets from polyvinylidene fluoride (PVDF) tubing connectors and durable peristaltic pump tubing. The length of the section is dependent on the distance between the different components.
    NOTE: Take special care when selecting tubing variety to ensure durability and reliability for long term experiments.
  3. Assemble the tubing set from three parts: a feed line, a waste line, and a sample line.
    1. Assemble the feed line using two tubing diameters (L/S 14 and L/S 13). Use the L/S 14 for the primary tubing that will span the distance from the bioreactor to the medium reservoir, and insert a short length of L/S 13 in the middle of the tubing length for the pump section using the appropriate PVDF adaptors.
    2. Use Standard PVDF hose-barbed tubing adaptors to join two pieces of L/S 14 tubing on either side of the shorter pump section (L/S 13) tubing.
      NOTE: Alternatively, the entire length of tubing could be smaller-diameter L/S 13 tubing, and the entire tubing length could be replaced when the pump-section integrity is in question.
  4. Assemble the second tubing section for waste removal using larger diameter (L/S 16) tubing and insert a short section of L/S 14 tubing in the middle for the pumping section. This is done in the same way as the feed line assembly using PVDF hose-barbed adaptors, or alternatively using a continuous length of the desired pump tubing size, and replacing as needed.
    NOTE: If the same pump and pump head are used for the feed circuit and the waste circuit, the pump section of the waste removal tubing lines must be a larger diameter than the pump section of the feeding line. This ensures that the removal pump rate is faster than the feed rate, and will avoid the potential for large changes in the culture volume, and reduce the risk of overflow.
  5. Assemble the final component of the tubing set: a sample collection assembly.
    1. This procedure describes a custom assembled sampling port system; alternatively, a sterile sampling port may be purchased from various vendors.
    2. Construct the set from three short (~ 6 cm) L/S 14 tubing lengths connected together with a T-type PVDF connector, and two small hose clamps on two of the tubing lengths.
    3. Attach a sterile gas filter for gas venting to one of the clamped lengths, and the other is used for connection to a sterile sampling syringe (sterile gas filters may need to be added in a bio-safety cabinet following sterilization as many sterile filters are not autoclavable).
    4. Connect the third end to a stainless steel sample connector attached on the lid of the bioreactor.
    5. Autoclave the sampling assembly while connected to the bioreactor prior to beginning the experiment (refer to step 3 below).
    6. Use this assembly to collect sterile samples from the bioreactor as needed without disturbing the continuous feed process.

3. Autoclave All Materials

  1. Prepare all bioreactor components for autoclave sterilization.
  2. Collect individual components for sterilization. Medium and waste reservoirs (assembled from step 2.1.1), 250 ml spinner flask (assembled from 2.1.1), necessary modified lids (assembled from 2.1.3), outflow tube (described in 2.1.4), three tubing sets (assembled in sections 2.2 through2.5), outflow tube (as needed for continuous feeding).
    1. Assemble spinner flask with all components and be sure the outflow tube is firmly attached inside the spinner flask (described in section 2.1.4), and attach the custom assembled sampling port (section 2.5), or other sampling port to the top of the lid.
    2. Wrap the entire spinner flask assembly with autoclave wrap material, and autoclave indicating tape. Be sure that the wrap airtight. NOTE: Aluminum foil or similar material can be used to cover the individual ends of each access port in the bioreactor lid to preserve sterility of the connector/ends when unwrapping and when non-connected to the tubing sets inside the incubator.
    3. Assemble the modified lids of the medium and waste reservoirs and then attach to the respective glass bottles. Cover them using aluminum foil and autoclave indicating tape.
    4. Individually wrap the tubing sets (section 2.2 - 2.5) with autoclave wrap and tape to assist in final assembly. Aluminum foil or similar material can be used to wrap the individual ends of each tubing set to preserve sterility of the connector/ends when unwrapping.
    5. Autoclave all wrapped items using a standard dry autoclave cycle (e.g., “Gravity” setting with ~ 15 psi at 121 °C for 30 min or more).
      NOTE: Do not attach sterile filters prior to autoclaving unless they are confirmed to be autoclave safe.

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.

  1. For all conditions, culture and expand β-TC6 cells in standard adherent cultures (described by vendor) until sufficient cell quantities are obtained to seed the desired number of bioreactors.
    1. Assemble the continuous feeding bioreactor with outflow tube as shown in Figure 1 and described in section 2, and connect sampling port to sampling lid. NOTE: The outflow tube and sampling port assembly should be added to the bioreactor for continuous feeding prior to spheroid formation, and should be pulled up out of the culture medium until continuous feeding is started.
    2. Sterilize the modified SSB assembly by autoclave (described in section 3).
    3. Attach sterile vents (0.22 µm or smaller sterile filters) to the appropriate locations on the lids of the spinner flask, and medium and waste vessels.
      NOTE: This is important to prevent vapor lock, and to allow gas exchange between the incubator (5% CO2) environment and the bioreactor. The gas exchange is necessary to maintain the correct pH when using bicarbonate-buffered media.
  2. Collect the cells by gentle trypsinization using 0.25% (w/v) Trypsin- 0.53 mM EDTA solution, at room temperature aided by mechanical agitation for 2 - 3 min, and seed into bioreactors at a density of approximately 1.3 x 106 cells/ml in 200 ml culture medium.
    1. Move designated flasks out of the incubator and into a Bio-Safety cabinet.
      NOTE: All cell culture and manipulations should be done in a Bio-Safety cabinet using proper sterile technique.
    2. Remove culture medium from flasks by aspirating.
    3. Wash cells by pipetting 5 ml of phosphate buffered saline (without Ca++ or Mg++), into flask, and rinsing across the cells on the surface, and then aspirate the PBS.
    4. Add 3 ml of Trypsin-EDTA solution to each flask that will be collected (typically about 10x 175 cm2 T-flasks).
    5. Allow harvested cells to incubate at room temperature for 2 - 3 min in bio-safety cabinet.
    6. Agitate gently by hand to loosen cells from flask surface, and collect cells by adding 6 ml of culture medium (with serum) to the flask, and transfer the cells to a 50 ml tube. When one 50 ml tube is full, use another 50 ml tube until all of the needed cells have been collected (approximately one 50 ml tube will be needed for every 5 T-175 flasks collected).
    7. Gently centrifuge (approximately 50 x gravity) the collected cell suspensions to pellet the cells.
    8. Aspirate the remaining medium leaving the pellet intact.
    9. Collect all of the cells in a single tube by re-suspending the pellets in culture medium (containing serum) using a pipette, and transferring all pellets into the same tube.
    10. Count the cell density using a standard hemocytometer with trypan blue staining as described in the literature53.
    11. Add desired number of total cells to sterile SSB for each condition (1.3 x 106 cells/ml was the targeted starting cell densities for this demonstration).
    12. Add medium (with the desired glucose concentration, in this case we used medium glucose levels within the physiological range) to SSB using a pipette to reach the desired total culture volume (200 ml culture volume was used for these studies).
      NOTE: For these continuous fed SSB studies the cultures were seeded using a modified “low-glucose” version of the culture medium (100 mg/dl). This allowed the cultures to start at the desired target glucose concentration rather than starting with a “high-glucose” medium and waiting for the glucose consumption to bring the glucose levels down into the physiological range to begin feeding. All other medium for feeding in these studies used the standard “high-glucose” medium (500 mg/dl).
    13. Move SSB with cells to a stir plate inside a cell culture incubator.
  3. Culture cells in the bioreactors without feeding for 3 days at 37 °C, with 5% CO2, 100% relative humidity, and a stir rate of 70 rpm to allow spheroids to form.
    NOTE: No significant proliferation should be observed during the three day spheroid formation period.
  4. After spheroid formation, divide spheroids among bioreactors with desired culture conditions.

5. Continuous Feeding Culture and Adjusted Feed Rate

  1. Autoclave or gas sterilize all of the components, and assemble in a biological safety cabinet using sterile techniques (steps 2 - 4).
  2. After spheroid formation, remove the SSB from the incubator and assemble the components for the continuous feeding system in a biological safety cabinet.
    1. First fill the fresh medium reservoir with the desired culture medium, and then connect to one side of the feed line. Also ensure that the fresh medium reservoir is properly vented with a sterile filter.
    2. Connect one side of the feed line to the feed inlet port on the bioreactor in a sterile manner. A sterile filter should be installed between the feed line and the bioreactor inlet port to filter the medium before it enters the bioreactor.
    3. Connect the waste line to the bioreactor outflow tube and the medium waste reservoir, and ensure that the waste reservoir is properly vented with a sterile filter.
  3. Move the assembly out of the biological safety cabinet (this will probably require two people to carry all of the vessels and tubing), and put all components out for long-term culture.
    1. Put the SSB onto the stir plate inside of the incubator using the same culture parameters for spheroid formation (37 °C, 5% CO2, 100% humidity and 70 rpm). NOTE: It may be necessary to temporarily disconnect the tubing segments from the bioreactor if the lines need to run through holes in the side of the incubator rather than out through the gasket in the door. This can be done in an aseptic way by wrapping the ends of the tubing sets with aluminum foil prior to autoclaving (step 3), and waiting to attach the bioreactor side of the feed and waste tube sections until the bioreactor is inside the incubator. The tube lines can be put in place, and the aluminum foil can be removed inside the incubator and quickly attached to the bioreactor.
    2. Run remaining ends of tubing sets (the ones not connected to the bioreactor lid) out through the incubator access port (pass-through), or through a notch in the incubator door gasket.
    3. Outside of the incubator (in a nearby biosafety cabinet if possible), quickly connect the feed line to the fresh medium reservoir, and the waste line to the medium waste reservoir, and ensure that the waste reservoir is properly vented with a sterile filter. NOTE: To do this in an aseptic manner, remove the aluminum foil from the tubing and vessel connectors and connect to the respective vessels as quickly as possible (especially if this connection needs to be done outside of the biosafety cabinet).
    4. Put fresh medium reservoir (filled with desired feed medium) inside nearby mini-refrigerator. Check to ensure that the feed and waste lines are able to exit the incubator and enter the refrigerator without preventing the doors on each from closing. It is also critical that the feed lines are not pinched closed by the doors of either the refrigerator or the incubator.
      NOTE: The medium used for these studies was the standard high glucose (500 mg/dl) culture medium recommended by the vendor for this cell type. Any high glucose medium could be used based on the specific needs of the cell type being cultured. The glucose concentration of the feed medium should be reasonably higher (2x or more) than the desired concentration in the culture as the feeding system relies on increasing the feed rate of high glucose medium to maintain adequate glucose levels in the cultures while maintaining reasonable feed rates.
    5. Next, the waste medium reservoir can be put on the bench top outside the incubator in a convenient location.
    6. Next, place the “pump section” of the feed and waste lines into the pump head ensuring the proper orientation so that the feed lines will pump medium from the fresh medium reservoir into the SSB, and the waste lines will pump medium from the SSB and to the waste medium reservoir.
  4. Turn the pump on to the desired feed rate.
    1. Calculate the feed rate using the adjusted feed rate equation described in the literature3 and in the provided representative results section below.
    2. Use the most recent cell count information (procedure described in step 5) along with the growth prediction, and the medium glucose measurements to estimate the feed rate needed to maintain the desired glucose concentration in the culture.
      NOTE: The cell growth rates and glucose consumption rates will need to be obtained prior to doing these procedures.
    3. Set the pump speed based on the calculated feed rate.
  5. Repeat the feed rate calculation and adjust the pump speed for each sampling point (every three days for the described method).

6. Cell Counts, Viability, and Glucose Concentration Measurements

  1. Collect samples from each bioreactor every three days, or as desired.
  2. Take culture samples from continuously fed SSB cultures using the specialized sampling port described above.
    1. Leaving the continuously fed SSB inside the bioreactor, attach a sterile syringe (a 5 ml volume syringe was used for these studies) to un-filtered connection of the sampling port.
    2. Move the sampling tube down into the culture medium.
    3. Unclamp the tube section going to the syringe, and withdraw the desired total sample volume.
    4. Move the sampling tube up so that it is no longer submerged in the culture, and continue to withdraw the syringe until air is removed.
    5. Clamp the tube that feeds the syringe, and detach the syringe containing the sample, and set aside.
    6. Pre-load a second fresh sterile syringe with air, and attach to the sterile filter side of the sampling port.
    7. Unclamp the tube going to the syringe-filter side of the sampling port, and then purge the sampling port by gently expelling the air in the syringe through the sterile filter. This ensures that the sampling port will be clear of any residual medium.
    8. Re-clamp the tube going to the filter, disconnect the sterile syringe and discard it.
  3. Take the syringe that contains the cell sample from the culture, and expel it into a 10 ml tube. Sub-samples of known volumes may be taken directly from this tube.
  4. For cell counts, remove a known volume of cells and transfer to a micro-centrifuge tube, and gently centrifuge for 1 - 2 min (approximately 50 x gravity).
  5. Transfer the supernatant to a separate tube for glucose measurements, and re-suspend the pellet with the same volume of trypsin-EDTA solution (0.25% (w/v) trypsin, 0.53 mM EDTA), and count in triplicate using a standard hemocytometer with trypan blue staining as described in the literature53.
    1. Add medium (with serum) to the sample for dilution as necessary.
    2. Count the cells, and calculate the cell viability by recording live (unstained) cells, and dead (stained) cells independently (viability % = live cells / total cells).
  6. Measure medium glucose levels in triplicate using a blood glucose meter and single use test-strips.
    1. Take glucose measurements as described by the glucose meter instructions substituting the culture medium for blood.
    2. Dip the test strip in the medium to be tested (collected from count samples above), and repeat for the desired number of replicates (three replicates are recommended).
    3. Test both the fresh medium and waste medium for glucose concentrations.
      NOTE: For some meters, measurements lower than 20 mg/dl (detectable threshold of the meter), may be observed as an error in the meter output.

7. Spheroid Settling Rate Measurements

  1. To ensure that cell clusters are not being removed by the continuous feeding system, measure the settling rate of β-TC6 spheroids by observing their sedimentation in a large diameter plastic pipette.
  2. Culture β-TC6 cells in SSB bioreactors to form spheroids as described above.
  3. After 3 days of culture, take 30 ml samples of the spheroid suspension and place in a 50 ml tube.
  4. Gently pipet up and down using a wide diameter 25 ml pipette to distribute evenly in suspension, then stop pipetting with the suspension at a known level in the pipet (e.g., 20 ml mark), and then record the time for all of the spheroids to settle 5 cm.
  5. Repeat this procedure enough times to attain statistical confidence (usually n > 3).
    NOTE: For biological studies, a p value less than 0.05 is considered to be statistically significant when comparing measurements. The standard error of the mean was used for reporting errors, and the two tailed un-paired student-t test was used to compare conditions for the presented data.

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Results

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 ...

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Discussion

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....

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
β TC-6 CellsATCC, Manassas, VACRL-11506Mouse Insulinoma cell line (adherent cell type)
DPBS No Ca, No MgInvitrogen, Carlsbad, CA14190-144https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144
Dulbecco's Modified Eagles MediumInvitrogen, Carlsbad, CASee below for product numbers 
DMEM High Glucose (500 mM)Invitrogen, Carlsbad, CA11965-092http://www.lifetechnologies.com/order/catalog/product/11965092
DMEM Low Glucose (100 mM)Invitrogen, Carlsbad, CA11885-084http://www.lifetechnologies.com/order/catalog/product/11885084 (note that this medium already contains pyruvate)
L-glutamineInvitrogen, Carlsbad, CA25030081http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product
Sodium PyruvateInvitrogen, Carlsbad, CA11360070https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product
Heat Inactivated Porcine SerumGibco - Life Technologies10082147http://www.lifetechnologies.com/order/catalog/product/10082147
Trypsin-EDTAInvitrogen, Carlsbad, CA25200056https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product
T-150 Tissue Culture Treated FlasksCorning, Corning, NY430825http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences)
&categoryname=
NuAire Cell culture incubatorPrinceton, MNUS Autoflow. Any water-jacketed CO2 regulating cell culture incubator could be used.
CentrifugeSorvall RT 7 (Any similar benchtop centrifuge may be used)
RefrigeratorAny laboratory refrigerator could be used (a small table-top version was used for these studies)
1 L Glass BottleCorning, Corning, NY1395-1LAny vendor could be used. http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=1395-1L(Lifesciences)
&categoryname=
2 L Glass BottleCorning, Corning, NY1395-2LAny vendor could be used
250 ml stirred bioreactors Corning, Corning, NY4500-250http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences)
&categoryname=
Stir PlateFisher Scientific11-496-104AAny incubator safe stir-plate can be used, any vendor
Tissue Culture Dishes 100 mm DiameterNunc, Rochester, NY (Fisher Scientific)1256598 Any vendor could be used (ordered through Fisher Sci)
FALCON 50 ml Conical TubesFalcon, San Jose, CA1256598Any vendor could be used
Delran Plastic Used for Custom PartsMcMaster CarrVariousAny 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 lidsMcMaster CarrVariousAny vendor could be used. http://www.mcmaster.com/#standard-stainless-steel-tubing/=rjrd91
Custom Modified Delran Bioreactor Lids for Continuous FeedingCustom madeNot aware of any vendors producing a similar product
Custom Modified Glass Bottle Lids for Continuous feedingCustom madeSome vendors (e.g., Fischer Sci, Corning) make similar products in the links below.
Masterflex Digital Peristaltic PumpCole Parmer, Vernon Hills, ILEW-77919-25Any 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, ILsee linkAny vendor could be used. http://www.coleparmer.com/Category/Cole_Parmer_PVDF_Premium
_Luer_Fittings/55889
Pharmed BPT Tubing L/S 16Cole Parmer, Vernon Hills, ILWU-06508-16Any 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 14Cole Parmer, Vernon Hills, ILWU-06508-14Any 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 13Cole Parmer, Vernon Hills, ILWU-06508-13Any 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 0.22 µl sterile syringe filter (used for venting, and medium filtration)Fisher ScientificSLGP033RSAny vendor could be used
25 ml Graduated PipetteFisher Scientific13-678-11Any vendor could be used, and various sizes may be used
PipetterFisher Scientific13-681-15EAny vendor, or similar product could be used
HemocytometerFisher Scientific02-671-6Any vendor, or similar product could be used
Trypan BlueGibco - Life Technologies15250-061Any vendor, or similar product could be used. https://www.lifetechnologies.com/order/catalog/product/15250061
Inverted Light MicroscopeLeicaAny vendor, or similar product could be used
One Touch Ultra Blood Glucose MeterFisher Scientific22-029-293Any vendor, or similar product could be used (e.g., Bayer)
One Touch Ultra-StripsFisher Scientific22-029-292 Any vendor, or similar product could be used (e.g., Bayer)

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