JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Please note that all translations are automatically generated. Click here for the English version.
Bioengineering

Närings förordning genom kontinuerlig matning för storskalig utbyggnad av däggdjursceller i sfäroider

Published: September 25, 2016
doi:
10.3791/52224
, , , , , , , , ,
1Radiology,University of Minnesota, 2Medicine,University of Minnesota, 3School of Public Health,University of Minnesota, 4Surgery,University of Arizona

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

För att generera ett stort antal viabla och funktionella humana celler för transplantation, är reglering av odlingsbetingelsema absolut nödvändigt. Utarmning av näringsämnen, tillsammans med uppbyggnaden av metaboliskt avfall är viktiga bidragsgivare med åldrande och metaboliska förändringar som minskar kvaliteten på cellprodukten 1-3. Denna procedur visar en metod för att odla däggdjursceller i sfäroider med användning av en omrörd bioreaktor i kombination med en justerad hastighet perfusion matningssystem för att reglera glukos i ett fysiologiskt intervall 4 under hela varaktigheten av kulturen. För ändamålet av dessa studier, var det fysiologiska området definieras som mellan 100 och 200 mg / dl. Samma metoder kan användas för att reglera andra näringsämnen och metaboliska avfall såsom laktat.

Statiska kulturer i små volymer (1 – 30 ml) används typiskt i laboratoriemiljö för att upprätthålla och differentiera cellinjer för experimespecialist- ändamål. Cell passage utförs med komplett medium de ändringar som behövs med jämna mellanrum. Mest "konventionella" odlingsmedium har en hög glukoskoncentration (450 mg / dl för DMEM användes i dessa studier) för att möjliggöra mindre frekventa mediumbyten utan risk för närings begränsningar. Men denna sats matning metoden kräver fortfarande frekvent manipulation, introducerar variabilitet i cellmiljön, och ökar risken för kontaminering 5-9. Omrörd suspension bioreaktorer (SSB) ger bättre blandning och minskad hantering 3,10 – 20, men som statiska kulturer kräver manuella medel förändringar som bidrar till potentiellt skadliga fluktuationer i närings och avfallsproduktnivåer. Perfusion utfodring av SSB kulturer minskar dessa problem genom kontinuerlig infusion och avlägsnande av medium, men stora förändringar i halter av näringsämnen på grund av celltillväxt förblir ett problem. Användningen av en justerad utfodrings rate från beräkningar av närings användning baserat på beräknade behov cell kan ge den stabila cellmiljön som krävs för att optimera cellviabilitet och funktion 21-24.

Det finns en stor mängd litteratur som beskriver metoder för skal SSB kulturer av däggdjursceller specifikt för kultur och utbyggnad av pluripotenta celler 25-32, med andra fokuserade på holme (beta) celler 17,33,34, eller produktion av biologiska produkter 24, 35-38. Många av dessa undersökta celltyper kan odlas i sfäroida kulturer, och särskilda förfaranden för den celltyp som används bör optimeras innan genomföra en kontinuerlig matningssystem. I denna demonstration var en perfusion matningsmetod som används för att expandera en beta cellinje odlas som sfäroider i en omrörd bioreaktor 39-43. Den metod som beskrivs häri tillhandahåller enunderlätta genomförandet av utfodring ränteförändringar baserade på off-line glukosmätningar för att uppnå riktade odlingsbetingelser. Justering av matningshastigheten med denna metod för att upprätthålla en fysiologisk glukosnivån visas till ökningar cellutbyten. Däggdjursceller är beroende på en nyckelkod näringsämne, glukos, för energiproduktion, så användningen av denna cellinje representerar en modell för många odlade däggdjursceller 44. Dessutom har denna linje exemplifierar ytterligare komplexitet betaceller, som är känsliga för kroniskt höga nivåer av glukos 45. För denna studie var p-TC6 celler tilläts bilda sfäroider i odling för att approximera den genomsnittliga storleken på Langerhanska öar in vivo. Perfusionen bioreaktorsystem 17 – 19,21,46 med en matningshastighet justeras för att glukoskonsumtion, resulterade i upprätthållande av fysiologiska betingelser och högre utbyten cell utan förändringar i viabilitet.

Protocol

1. Cell Line och underhåll Skaffa p-TC6 celler (eller annan önskad vidhäftande däggdjurscellinje). Som förberedelse för studien, kultur, passage, och cryopreserve cellerna enligt leverantörens instruktioner. 2. Montera kontinuerlig matning System OBS! Kontinuerlig matning systemdesign i metoden nedan baserades på liknande system som beskrivs i litteraturen 17 – 19,21,47 – 49. Montering …

Representative Results

Medium glukosnivåer och fluktuationer Begränsa Cell Expansion i Standard SSB kulturer Glukosnivåerna varierar i statiska kulturer och SSB kulturer över hela odlingsperioden 3. Dessa svängningar öka med ökande cellantal under 21-dagars odlingsperiod och var nästan identiska i både statiska och SSB kulturer. Dessa observationer presenteras i vår tidigare publikation 3. Glukosnivåerna kan vara super-fysiologiska under hela odlingsperioden för b…

Discussion

Generera däggdjurscellprodukter för framställning av biologiska agens och cellterapi kräver kultur och övervakning av däggdjursceller i stor skala 55-58. Vidare har dessa applikationer kräver definierade och validerade odlingsbetingelser. Helt enkelt öka volymen av celler med användning av forsknings teknik kommer inte uppfyller alla dessa krav. Manuella ändringar medel orsakar svängningar i näringsämnen och ackumulering av avfallsprodukter minskar cellernas kvalitet, lönsa…

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.

Materials

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)
DOWNLOAD MATERIALS LIST

References

  1. Reuveny, S., Velez, D., Macmillan, J. D., Miller, L. Factors affecting cell growth and monoclonal antibody production in stirred reactors. J. Immunol. Methods. 86 (1), 53-59 (1986).
  2. Tarleton, R. L., Beyer, A. M. Medium-scale production and purification of monoclonal antibodies in protein-free medium. Biotechniques. 11 (5), 590-593 (1991).
  3. Weegman, B. P., et al. Nutrient regulation by continuous feeding removes limitations on cell yield in the large-scale expansion of Mammalian cell spheroids. PLoS One. 8 (10), e76611 (2013).
  4. Klueh, U., et al. Continuous glucose monitoring in normal mice and mice with prediabetes and diabetes. Diabetes Technol. Ther. 8 (3), 402-412 (2006).
  5. Hay, R. J. Operator-induced contamination in cell culture systems. Dev. Biol. Stand. 75, 193-204 (1991).
  6. Dazey, B., Duchez, P., Letellier, C., Vezon, G., Ivanovic, Z. Cord blood processing by using a standard manual technique and automated closed system “Sepax” (Kit CS-530). Stem Cells Dev. 14 (1), 6-10 (2005).
  7. Gastens, M. H., et al. Good manufacturing practice-compliant expansion of marrow-derived stem and progenitor cells for cell therapy. Cell Transplant. 16 (7), 685-696 (2007).
  8. Naing, M. W., Williams, D. J. Three-dimensional culture and bioreactors for cellular therapies. Cytotherapy. 13 (4), 391-399 (2011).
  9. Stacey, G. N. Cell culture contamination. Cancer Cell Culture. , 79-91 (2011).
  10. Zur Nieden, I. N., Cormier, J. T., Rancourt, D. E., Kallos, M. S. Embryonic stem cells remain highly pluripotent following long term expansion as aggregates in suspension bioreactors. J. Biotechnol. 129 (3), 421-432 (2007).
  11. Kehoe, D. E., Jing, D., Lock, L. T., Tzanakakis, E. S. Scalable stirred-suspension bioreactor culture of human pluripotent stem cells. Tissue Eng. Part A. 16 (2), 405-421 (2010).
  12. Krawetz, R., et al. Large-scale expansion of pluripotent human embryonic stem cells in stirred-suspension bioreactors. Tissue Eng. Part C. Methods. 16 (4), 573-582 (2010).
  13. Shafa, M., et al. Expansion and long-term maintenance of induced pluripotent stem cells in stirred suspension bioreactors. J. Tissue Eng. Regen. Med. 6 (6), 462-472 (2012).
  14. Oh, S. K. W., et al. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res. 2 (3), 219-230 (2009).
  15. Olmer, R., et al. Suspension culture of human pluripotent stem cells in controlled, stirred bioreactors. Tissue Eng. Part C. Methods. 18 (10), 772-784 (2012).
  16. Baptista, R. P., Da Fluri, ., Zandstra, P. W. High density continuous production of murine pluripotent cells in an acoustic perfused bioreactor at different oxygen concentrations. Biotechnol. Bioeng. 110 (2), 648-655 (2013).
  17. Papas, K. K. . Characterization of the metabolic and secretory behavior of suspended free and entrapped ART-20 spheroids in fed-batch and perfusion cultures [dissertation]. , (1992).
  18. Papas, K. K., Constantinidis, I., Sambanis, A. Cultivation of recombinant, insulin-secreting AtT-20 cells as free and entrapped spheroids. Cytotechnology. 13 (1), 1-12 (1993).
  19. Sambanis, A., Papas, K. K., Flanders, P. C., Long, R. C., Kang, H., Constantinidis, I. Towards the development of a bioartificial pancreas: immunoisolation and NMR monitoring of mouse insulinomas. Cytotechnology. 15 (1-3), 351-363 (1994).
  20. Sharma, S., Raju, R., Sui, S., Hu, W. -. S. Stem cell culture engineering – process scale up and beyond. Biotechnol. J. 6 (11), 1317-1329 (2011).
  21. Papas, K. K., Long, R. C., Constantinidis, I., Sambanis, A. Role of ATP and Pi in the mechanism of insulin secretion in the mouse insulinoma betaTC3 cell line. Biochem. J. 326 (Pt 3), 807-814 (1997).
  22. Papas, K. K., Long, R. C., Sambanis, A., Constantinidis, I. Development of a bioartificial pancreas: I. long-term propagation and basal and induced secretion from entrapped betaTC3 cell cultures. Biotechnol. Bioeng. 66 (4), 219-230 (1999).
  23. Papas, K. K., Long, R. C., Sambanis, A., Constantinidis, I. Development of a bioartificial pancreas: II. Effects of oxygen on long-term entrapped betaTC3 cell cultures. Biotechnol. Bioeng. 66 (4), 231-237 (1999).
  24. Hu, W. S. Cell culture process monitoring and control-a key to process optimization. Cytotechnology. 14 (3), 155-156 (1994).
  25. Alfred, R., et al. Efficient suspension bioreactor expansion of murine embryonic stem cells on microcarriers in serum-free medium. Biotechnol. Prog. 27 (3), 811-823 (2011).
  26. Cormier, J. T., zur Nieden, N. I., Rancourt, D. E., Kallos, M. S. Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors. Tissue Eng. 12 (11), 3233-3245 (2006).
  27. Dang, S. M., Zandstra, P. W. Scalable production of embryonic stem cell-derived cells. Methods Mol. Biol. 290 (1), 353-364 (2005).
  28. Elseberg, C. L., et al. Microcarrier-based expansion process for hMSCs with high vitality and undifferentiated characteristics. Int. J. Artif. Organs. 35 (2), 93-107 (2012).
  29. Kallos, M. S., Behie, L. A. Inoculation and growth conditions for high-cell-density expansion of mammalian neural stem cells in suspension bioreactors. Biotechnol. Bioeng. 63 (4), 473-483 (1999).
  30. Kehoe, D. E., Lock, L. T., Parikh, A., Tzanakakis, E. S. Propagation of embryonic stem cells in stirred suspension without serum. Biotechnol. Prog. 24 (6), 1342-1352 (2008).
  31. Kirouac, D. C., Zandstra, P. W. The systematic production of cells for cell therapies. Cell Stem Cell. 3 (4), 369-381 (2008).
  32. Serra, M., et al. Stirred bioreactors for the expansion of adult pancreatic stem cells. Ann. Anat. 191 (1), 104-115 (2009).
  33. Chawla, M., Bodnar, C. A., Sen, A., Kallos, M. S., Behie, L. A. Production of islet-like structures from neonatal porcine pancreatic tissue in suspension bioreactors. Biotechnol. Prog. 22 (2), 561-567 (2006).
  34. Weegman, B. P., et al. Temperature profiles of different cooling methods in porcine pancreas procurement. Xenotransplantation. , (2014).
  35. Cruz, H. J., Moreira, J. L., Carrondo, M. J. Metabolic shifts by nutrient manipulation in continuous cultures of BHK cells. Biotechnol. Bioeng. 66 (2), 104-113 (1999).
  36. Dowd, J. E., Jubb, A., Kwok, K. E., Piret, J. M. Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates. Cytotechnology. 42 (1), 35-45 (2003).
  37. Goudar, C., Biener, R., Zhang, C., Michaels, J., Piret, J., Konstantinov, K. Towards industrial application of quasi real-time metabolic flux analysis for mammalian cell culture. Cell Culture Engineering. 101, 99-118 (2006).
  38. Hu, W. S., Piret, J. M. Mammalian cell culture processes. Curr. Opin. Biotechnol. 3 (2), 110-114 (1992).
  39. Knaack, D., et al. Clonal insulinoma cell line that stably maintains correct glucose responsiveness. Diabetes. 43 (12), 1413-1417 (1994).
  40. Poitout, V., Stout, L. E., Armstrong, M. B., Walseth, T. F., Sorenson, R. L., Robertson, R. P. Morphological and functional characterization of beta TC-6 cells–an insulin-secreting cell line derived from transgenic mice. Diabetes. 44 (3), 306-313 (1995).
  41. Poitout, V., Olson, L. K., Robertson, R. P. Insulin-secreting cell lines: classification, characteristics and potential applications. Diabetes Metab. 22 (1), 7-14 (1996).
  42. Suzuki, R., et al. Cyotomedical therapy for insulinopenic diabetes using microencapsulated pancreatic beta cell lines. Life Sci. 71 (15), 1717-1729 (2002).
  43. Skelin, M., Rupnik, M., Cencic, A. Pancreatic beta cell lines and their applications in diabetes mellitus research. ALTEX. 27 (2), 105-113 (2010).
  44. Masters, J. R., Stacey, G. N. Changing medium and passaging cell lines. Nat. Protoc. 2 (9), 2276-2284 (2007).
  45. Murdoch, T. B., McGhee-Wilson, D., Shapiro, A. M. J., Lakey, J. R. T. Methods of human islet culture for transplantation. Cell Transplant. 13 (6), 605-617 (2004).
  46. Woodside, S. M., Bowen, B. D., Piret, J. M. Mammalian cell retention devices for stirred perfusion bioreactors. Cytotechnology. 28 (1-3), 163-175 (1998).
  47. Serra, M., et al. Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control. J. Biotechnol. 148 (4), 208-215 (2010).
  48. Gálvez, J., Lecina, M., Solà, C., Cairó, J., Gòdia, F. Optimization of HEK-293S cell cultures for the production of adenoviral vectors in bioreactors using on-line OUR measurements. J. Biotechnol. 157 (1), 214-222 (2012).
  49. Trabelsi, K., Majoul, S., Rourou, S., Kallel, H. Development of a measles vaccine production process in MRC-5 cells grown on Cytodex1 microcarriers and in a stirred bioreactor. Appl. Microbiol. Biotechnol. 93 (3), 1031-1040 (2012).
  50. Liu, H., et al. A high-yield and scaleable adenovirus vector production process based on high density perfusion culture of HEK 293 cells as suspended aggregates. J. Biosci. Bioeng. 107 (5), 524-529 (2009).
  51. Zhi, Z., Liu, B., Jones, P. M., Pickup, J. C. Polysaccharide multilayer nanoencapsulation of insulin-producing beta-cells grown as pseudoislets for potential cellular delivery of insulin. Biomacromolecules. 11 (3), 610-616 (2010).
  52. Lock, L. T., Laychock, S. G., Tzanakakis, E. S. Pseudoislets in stirred-suspension culture exhibit enhanced cell survival, propagation and insulin secretion. J. Biotechnol. 151 (3), 278-286 (2011).
  53. Marchenko, S., Flanagan, L. Counting human neural stem cells. J. Vis. Exp. (7), e262 (2007).
  54. Campos, C. Chronic hyperglycemia and glucose toxicity: pathology and clinical sequelae. Postgrad. Med. 124 (6), 90-97 (2012).
  55. Eve, D. J., Fillmore, R., Borlongan, C. V., Sanberg, P. R. Stem cells have the potential to rejuvenate regenerative medicine research. Med. Sci. Monit. 16 (10), RA197-RA217 (2010).
  56. Hsiao, L. -. C., Carr, C., Chang, K. -. C., Lin, S. -. Z., Clarke, K. Review Article: Stem Cell-based Therapy for Ischemic Heart Disease. Cell Transplant. , (2012).
  57. Oldershaw, R. A. Cell sources for the regeneration of articular cartilage: the past, the horizon and the future. Int. J. Exp. Pathol. 93 (6), 389-400 (2012).
  58. De Coppi, P. Regenerative medicine for congenital malformations. J. Pediatr. Surg. 48 (2), 273-280 (2013).
  59. Tziampazis, E., Sambanis, A. Modeling of cell culture processes. Cytotechnology. 14 (3), 191-204 (1994).
  60. Sidoli, F. R., Mantalaris, A., Asprey, S. P. Modelling of Mammalian cells and cell culture processes. Cytotechnology. 44 (1-2), 27-46 (2004).
  61. Yim, R. Administrative and research policies required to bring cellular therapies from the research laboratory to the patient’s bedside. Transfusion. 45, 144S-158S (2005).
  62. Fink, D. W. FDA regulation of stem cell-based products. Science. 324 (5935), 1662-1663 (2009).
  63. Moos, M. Stem-cell-derived products: an FDA update. Trends Pharmacol. Sci. 29 (12), 591-593 (2008).

Tags

Nutrient RegulationContinuous FeedingLarge-scale Cell ExpansionSpheroidsBioreactorPeristaltic PumpMedium ReservoirWaste ReservoirPerfusion TubingCell CultureCellular TherapyNutrient ControlGas ExchangeCell DensityCell YieldCell Loss

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Weegman, B. P., Essawy, A., Nash, P., Carlson, A. L., Voltzke, K. J., Geng, Z., Jahani, M., Becker, B. B., Papas, K. K., Firpo, M. T. Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids. J. Vis. Exp. (115), e52224, doi:10.3791/52224 (2016).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image