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Modes of culture for high cell densities - Workforce Solutions

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					Modes of culture for high cell
                     densities
            Chapter 10 ‘The Basics’
What is a Batch Culture?

l Cells are inoculated –
  culture left for several
  days – until final
  density is reached
l Nothing is added or
  removed during culture
l Substrates get used up
  and products are
  secreted from cells
Heterogeneous system
Detrimental environment for cell growth


- Depletion of an essential nutrient

- Accumulation of an inhibitor

- Complete coverage of available growth
  surface
What is Fed-batch culture?

l Cell growth – nutrient supply or removal of by-
  products

l Cell yield will reach high densities

l In open system/Fed-batch culture – involves
  controlled nutrient feeding

l Partial media changes at regular intervals
What is a Continuous Culture?

l Open system – continuous feed of medium and
  removal of ‘spent’ medium
l Cell growth - longer period in CC>BC
Types of Continuous Culture


lChemostat culture – Cells and spent
 medium are continuously removed

- State of culture is dependent upon flow rate of
  fresh medium
Types of Continuous Culture

lPerfusion culture – Cells are retained in
  fermenter
- Medium is pumped continuously
- Cells are retained
- Becoming popular for large-scale production
- Attains high cell density
- Cell separator
Cell immobilization

lImmobilization of cells on or inside particles

lAllows attachment of cells to solid surface

lAnchorage dependent cells

lEntrapment of cells in small beads
What are Microcarriers?
l Microcarriers are
  microscopic particles
  (diameter = 200 μm)

l Maintained in
  suspension in liquid
  medium

l Dextran, Collagen and
  Plastic
    Characteristics of Microcarriers

lSmall – to maximize the available surface area
 for cell growth
lLight – to allow easy suspension in culture
 medium
lTransparent – to allow easy observation of
 cell attachment and growth
lCharged – to allow cell attachment onto
 surface
Porous microcarriers

lDextran microcarriers (example: Cytodex)
   are microporous
 - Pore size is not sufficient to allow cells to
   colonize the interior of beads
lGelatin microcarriers (example: Cultispher)
   are macroporous
- Increased surface area for attachment
- Interior environment to protect cells against
   adverse shear forces
Extraction of cells from Microcarriers

lDetachment by either trypsin or collagenase
 treatment

lDetached cells separated by sieving through a
 nylon mesh
Immobilization of nonanchorage-dependent
cells


lProtection against mechanical stress

lEase of continuous operation

lIsolation of products
Immobilization of nonanchorage-dependent
cells
lCell entrapment –
- Mixing and agitating suspension of cells in
  warm agarose with hydrophobic liquid –
  paraffin oil
- Forms solid beads of agarose (100-200 μm)
  containing suspended cells
- Secreted cellular products – separated from
  entrapped cells
Immobilization of nonanchorage-dependent
cells - Encapsulation
l Cells – enclosed in
  semipermeable membrane
l Cells + sodium alginate
  drip into calcium alginate
l Polylysine creates an outer
  semipermeable membrane
l Monoclonal antibodies
  accumulate inside the bead
  matrix – can be extracted
  easily
l   This project is funded by a grant awarded under the President’s Community Based Job Training Grant as 
    implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60).  
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    political affiliation or belief; and
l   against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 
    (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in 
    the United States, or his or her participation in any WIA Title I-financially assisted program or activity.
Disclaimer
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  Administration.  The solution was created by the grantee and does 
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