Presentation is loading. Please wait.

Presentation is loading. Please wait.

Bioreactor design Issues for cell cultures. Cell Culture - An engineering perspective.

Similar presentations


Presentation on theme: "Bioreactor design Issues for cell cultures. Cell Culture - An engineering perspective."— Presentation transcript:

1 Bioreactor design Issues for cell cultures

2 Cell Culture - An engineering perspective

3 by Genentech, Corporate Communication A Fermenter / Bioreactor And Its Parts

4 Single System for Anchorage-Dependent and Suspension Cultures New Brunswick Scientific Company

5 BioFlo® Pro Customizable Cell Culture Bioreactors

6 Fig. 2. Influenza production plant (6000 liter vessel for cultivating Vero cells on Cytodex™). Courtesy of Baxter Biosciences.

7 Nutrient Considerations Environment Considerations Common Culturing Systems Examples Type of cultures

8 Suspension cultures Anchorage dependent cultures monolayer

9 Bioreactor: Advantages Controlled environment: 1.Mixing 2.pH 3.Dissolved oxygen 4.Temperature

10 pH probe 1.Steam sterilizable 2.Combination electrode 1.Two major types a.Galvanic b.Polargraphic Dissolved oxygen probe

11 Galvanic and Polargraphic Probes Cathode 0.5 O 2 + H 2 O + 2e -  2OH - Pt Anode (galvanic) Pb  Pb e - Anode (polargraphic) Ag + Cl -  AgCl + e -

12 Two major classes serum supplemented serum-free (or low serum)

13 Environment considerations - nutrient supply - mixing - oxygen supply - pH - carbon dioxide - NaHCO or NaOH 3 - temperature - waste accumulation - lactate - ammonia

14

15 Kolmogorov length scale (microns) Relative net growth rate versus Kolmogorov eddy length scale for FS-4 cultures with 0.2 g/l microcarriers Relative specific growth rate

16 Nucleic acid synthesis glutamine glutamate glycine alanineasparatate TCA cycle citratemalate oxaloacetate phosphoenolpyruvate glycolysis glucose pyruvate lactate -ketoglutarate    Schematic representation of some of the interrelationships of glucose an glutamine metabolism in mammalian cells

17

18

19

20

21

22

23

24 p p h Ie h

25 riri roro rcrc [O 2 ] [O 2 ] c riri roro rcrc fibre [O 2 ] – oxygen conc [O 2 ] c – critical oxygen conc

26

27

28 CellMax® artificial capillary cell culture system®

29 FiberCell Systems, Inc.

30 Cells grow on and around hollow fibers. ♦ Fiber geometry is optimized for both adherent and suspension cell types. ♦ Small molecules such as lactate, and glucose can easily cross the fiber. ♦ Large molecules such as mono clonal antibodies and proteins are retained and concentrated in the small volume of the extra capillary space.

31

32

33 Source: GE Healthcare – Microcarrier Cell Culture: Principles and Methods

34

35 Typical cell growth on microcarriers

36

37 FibraCel ® Disks A Solid Support Growth Material for Mammalian, Animal & Insect Cells Table 1 - Summary of cells commonly used successfully on FibraCel disks

38 Table 1 - Summary of cells commonly used successfully on FibraCel disks HybridomaAnchorage-DependentInsect DA A 127A GAMMA 67-9-B 3T3, COS, Human Osteosarcoma MRC-5, BHK, VERO CHO, rCHO-tPA rCHO – Hep B Surface Antigen HEK 293, rHEK 293 rC127 – Hep B Surface Antigen Normal Human Fibroblasts Stroma Hepatocytes Tn-368 SF9 rSF9 Hi-5 FibraCel® Disks

39 Table 1 - Summary of cells commonly used successfully on FibraCel disks FibraCel® Disks Yes Autoclavable YesCytotoxicity tested YesBioburden tested YesEndotoxin tested 3 x 10 5 cells/mL final volumeRequired inoculum 6 mmDisk diameter 1200 cm 2 Surface Area per gram Specifications

40 Perfusion system - to provide fresh nutrient - to remove waste (especially toxic byproducts - mechanical signal

41 Fig. 1 Schematic diagram of the perfusion–bleeding culture system. The settler consists of a cylinder part and a cone part. Dimensions of the settler: height of the cylinder, 5.5 cm; height of the cone, 5.5 cm; internal diameter (i.d.) of the cylinder, 5 cm; i.d. of pipes number 1 and number 3, 3 mm; i.d. of pipe number 2, 5 mm. Pipe number 1 is connected to the settler in the middle part of the cylinder (Z.-Y. Wen and F. Chen, Applied Microbiology and Biotechnology, 57: 316 – 322, 2001)

42 S. Zhang, A. Handa-Corrigan,and R.E. Spier, BIOTECHNOLOGY AND BIOENGINEERING, VOL. 41, NO. 7, MARCH 25, 1993 Figure 1. Schematic diagram of the perfusion culture system.

43

44

45

46 Large 3-D Cellular Aggregates BHK-21 Cell Culture Forms 2,000  m 3- D Cellular Aggregates within Two Days

47 Questions?

48 Transport in a Grooved Perfusion Flat-Bed Bioreactor for Cell Therapy Applications Marc Horner, William M. Miller, J. M. Ottino, and E. Terry Papoutsakis Biotechnol Prog 1998 Sep-Oct;14(5):689-98

49 Figure 1. Model of the perfusion chamber, a flat-bed bioreactor in which a series of 190 grooves at the chamber bottom (shown in figure) retains cells in the presence of constant medium perfusion. This is a closed system, with no headspace when the lid is placed on top. Medium flows in the z-direction across the chamber. y  and z  represent the local coordinate system in a cavity.

50 A Microfabricated Array Bioreactor for Perfusion 3-D Liver Culture Mark J. Powers et. al Bioengineering & Biotechnology, 2002, 78:257-69

51

52

53 Examples

54

55 Cell-polymer implants Isolated chondrocytes Cartilage biopsy In vitro tissue culture Polymer scaffold Petri dish Bioreactor In vivo implantation Implant Proposed Therapy

56

57 Fig 3. Effects of scaffold thickness and implant cultivation time on cell growth rate

58 6 2 4 Doubling time (days) Fig. 4 Effect of scaffold thickness on cell doubling time Scaffold thickness

59

60 Hi Me Lo Hi Me Lo Cell Density Petri dishBioreactor Doubling time (days) Fig. 6 Effects of Cell density on cell doubling time

61 Gas Exchange is Essential for Bioreactor Cultivation of Tissue Engineered Cartilage Bojana Obradovic, Rebecca L. Carrier, Gordana Vunjak- Novakovic, Lisa E. Freed Biotechnology and Bioengineering, 63: 197–205, 1999.

62 Figure 1. Model system. Isolated primary chondrocytes are seeded onto fibrous, biodegradable PGA scaffolds and cultured in vitro for 5 weeks in rotating bioreactors under different conditions of gas and medium exchange.

63 Group 1 (control) — regular medium replacement (50% v/v, 3 times per week), continuous gas exchange Group 2 (infrequent gassing) — regular medium replacement (50% v/v, 3 times a week), periodic gas exchange (3 times per week for 5 h, after medium replacement) Group 3 (no gassing) — regular medium replacement (50% v/v, 3 times per week), no gas exchange Group 4 (infrequent feeding) — Infrequent medium replacement (50% v/v, once per week), continuous gas exchange

64 Table II. Biochemical compositions of cell–polymer constructs.

65 Table III. Cell metabolism in cell–polymer constructs.

66 Comparison of Chondrogensis in Static and Perfused Bioreactor Culture David Pazzano,† Kathi A. Mercier,†, | John M. Moran,†,‡ Stephen S. Fong,†,‡ David D. DiBiasio,‡ Jill X. Rulfs,§ Sean S. Kohles, | and Lawrence J. Bonassar*,† Biotechnol Prog. 16(5):893-6 (2000)

67 Figure 1. Schematic representation of the perfusion bioreactor system assembly.

68

69

70

71 Figure 3. (A) Static sample at 2 weeks stained with safranin-O/fast green revealed light staining and no discernible orientation (400, bar ) 10 Ì m). (B) Bioreactor sample at 2 weeks stained with safranin-O/fast green (400, bar ) 10 Ì m). Intense staining was observed, as well as alignment of cells in the direction of media flow. A B

72 Cardiac Tissue Engineering: Cell Seeding, Cultivation Parameters, and Tissue Construct Characterization Rebecca L. Carrier, Maria Papadaki, Maria Rupnick, Frederick J. Schoen, Nenad Bursac,5 Robert Langer, Lisa E. Freed, Gordana Vunjak-Novakovic Biotechnol Bioeng. 64(5):580-9 (1999)

73 Figure 1. Effect of seeding vessel on the cellularity and metabolic activity of 3- day constructs. (a) DNA content ( m g/construct) (*) significantly greater than mixed flask group, p < 0.05 (n 4 4). (b) Medium LDH content (total U over 3 days of seeding) (*) significantly greater than all other groups, p < 0.05 (n 4 4). (c) Tetrazolium conversion (MTT assay OD units/mg DNA) (*) significantly greater than all other groups, p < 0.05 (n 4 4).

74 Figure 4. Cardiac-specific features: Constructs cultured for 1 week in a HARV (a, c, d) or a flask mixed at 50 rpm (b) and immunohistochemically labeled for (a) muscle desmin, (b) cardiac myosin, (c) cardiac troponin-T, and (d) sarcomeric tropomyosin. The arrow denotes a polymer fiber. (e) Transmission electron photomicrograph from a cardiac construct cultured for 1 week in a HARV demonstrating several adjacent cardiac myocytes with intercellular desmosome-like junctions (small arrows), myofibrils with sarcomeric organization highlighted by z lines (broad arrow), and compact mitochondria (open arrow). The nucleus of one cell is designated by the asterisk. Scale bars are 25 m m in a–d and 2 m m in e (original magnification 12,000).

75 Questions?

76 Extras

77

78

79 Typical oxygen consumption rate

80 “Protection” Property of Pluronic F-68

81

82 Photograph of BHK-21 cells om CMSM-GG microcarriers (200X)

83

84 Photograph of hepa-1,6 cells on magnetite microcarriers cultured in a MSFB bioreactor (400X)

85 Photograph of hepa-1,6 cells on magnetite microcarriers cultured in a MSFB bioreactor (400X)

86

87


Download ppt "Bioreactor design Issues for cell cultures. Cell Culture - An engineering perspective."

Similar presentations


Ads by Google