L1 cell culture by lanyuehua


									ABE / BME / CHEE 481B, 581B

      Dr. Mark Riley
      Ag. and Biosystems Engineering
      Spring 2008
      Lecture 1: Cell culture
Format of class

   1) Design of production of pharmaceutical
   2) Design of tissue engineered devices
Overview of lecture 1
   Overview of the cell culture approach
   Cell physiology and structure
   Products from cell culture
   Cell classification and isolation
       Cell adhesion to surfaces
       Transformed cells
       Primary cells and cell types
   Cellular metabolic requirements
Learning objectives for lecture #1

   Introduction to physiology and nutritional
    requirements of animal cells
   Common tests performed to assess cell
    function and suitability of their

   Read: Palsson, Chp 1 & 10
A “typical” pharma company
   Merck
       Basic science
           Discovery of new compounds which may have therapeutic
       Research
           Assessment of how the compound acts
       Development
           Determines ways to make the compound
       Pilot plant
           Moderate scale production
               Develop methods, produce material for clinical trials
       Production
           Large scale manufacture
Challenge for the day

   You are a new development engineer at the
    pharmaceutical firm, Merck, in the Biochemical
    research division
   A company has approached your boss with their
    new product which they claim provides an
    excellent material for growth of any animal cell
   Your task is to design experiments to assess if
    this would work for your group.
    Challenge for the day

   Assess if a cell culture is functioning well
       Identify cell type
           Anch / non-anch
           Morphology (shape)
       Physiology, growth rate, nutrient uptake,
        product formation
    What is cell culture?
   The maintenance of individual cells
    that have been harvested from an
    animal in a viable and metabolically
    active state.
   If the cells are fed a supply of
    nutrients and growth factors, they
    can remain active for days to years.
   The culture process allows cells to               Epithelial cells in
    act as individual units capable of                culture, stained for
                                                      keratin (red) and
    reproducing, metabolising, and                    DNA (green)
    responding to their environment.

    Cell culture
   One of the earliest human
    cell lines, descended from
    Henrietta Lacks, who died of
    the cancer that those cells
    originated from, the
    cultured HeLa cells shown
    here have been stained with
    Hoechst turning their nuclei

     Cell culture applications
   Product formation
        using animal cells for pharmaceutical development as animal cells can
         perform the necessary post-translational processing (protein folding and
         glycosylation) necessary to avoid initial of an immune response in the inject
   Toxicity testing
        cell culture methods can speed the evaluation process, provide more
         mechanistic understanding, reduce cost, and reduce animal rights concerns.
   Tissue engineering
        involves replacing the function of many cells or a tissue within an whole
        artificial liver, artificial skin
Products generated by
animal cell culture include:

   1) Enzymes - blood clotting compounds - Factor VII,
    Factor VIII, Factor X
   2) Hormones- erythropoetin - stimulates blood cell
    production (cures anemia)
   3) Viral vaccines - mumps, measles, rubella (MMR);
    rabies; influenza; polio; smallpox
   4) Antibodies - used as diagnostic and therapeutic
   5) Cytokines - Immunoregulators
       Interferons
       Interleukins
       Tumor necrosis factor (TNF)
    Viral vaccines
   Production of vaccines for viruses often uses cell cultures
   Global vaccine market $3B
   A vaccine contains antigenic components related to a
   Administration of vaccine leads to activation of the host
    immune system (both humoral and cellular)
   Prepares the immune system to respond to further challenges
    of the same antigen

                                              Human rhinovirus
    Viral vaccines

   Hepatitis A viral vaccine - grown in human
   Varicella-zoster vaccine - grown in a variety of
    human cell cultures
   Polio virus vaccine - grown in monkey kidney cells
   Rubella (for MMR) vaccine - grown in a variety of
    human cell cultures
   Production of viruses
                                                                       Replication of
                                           Infection                   DNA / RNA



                             Lysis and spread of
                             virus particles

A virus attaches to the host cell and enters endocytosis.
The capsid protein dissociates and the viral RNA is transported to the nucleus.
In the nucleus, the viral polymerase complexes transcribe and replicate the RNA.
Viral mRNAs migrate to cytoplasm where they are translated into protein.
Then the newly synthesized virions bud from infected cell.
Technical challenges
   Selecting susceptible cell
   Timing of infection
   Purification
   Attenuation - the process of eliminating (or
    greatly reducing) the virulence of a pathogen
       chemical treatment, heat inactivation, growth in a
        non-native host
       small chance that the virus could revert to infective
           chemical treatment, heat inactivation
           must be 100% effective
    Cell physiology

   Relevant components for our discussion
       Membrane
       Cytoskeleton
           Actin
           Microtubules
      Membrane proteins can
      associate with the lipid bilayer
                    Outside cell


                     Inside cell

Animal cell membrane has a net negative charge due to phosphate groups (PO3-)
on lipids (phospholipids) and cell surface proteins (which often are
phosphorylated – have an added phosphate, or are glycosylated (sugar residues)).
  Adherence to surfaces –
  focal adhesions
                                                    Vinculin is located only at focal
                                                    adhesions, which is also where
                                                         bundles of actin filaments
                                                           terminate at the plasma
                            Actin filament                               membrane.

            Vinculin                                       INSIDE
                          Actinin, talin
                                 Integrins                 OUTSIDE
Extracellular                                              CELL

                       Adapted from Alberts, et al., Molecular Biology of the Cell, 4th Ed.

   Actin filaments
   Microtubules
   Intermediate filaments
Twentieth Place, 2004 Competition, Albert Tousson Department of Cell
Biology, University of Alabama at Birmingham
Cultured baby hamster kidney cells (1500x) Fluorescence
    Actin filaments
   Two stranded helical polymers of the
    protein actin.
   Flexible structures of diameter 5-9 nm.
   Most highly organized on the periphery of
    the cell (cortical actin)
       Also found in stress fibers that cross the
   Found in 2 forms:
       G-actin – globular protein
       F-actin – filaments
           Formed by polymerization of G-actin
   F-actin structures are dynamic with a fast-
    growing “+” end.
Polymerization of actin

   Regulated by several proteins that modulate the
    rate of growth and lifetime
       Change as a function of the cell activity
           Growing, moving, or quiescent (confluent monolayer)
       Mean filament lifetime changes significantly
           40 minutes (quiescent cells)
               70% of actin is polymerized
           8 minutes (sub-confluent, motile cells)
               40% of actin is polymerized
   Hollow cylinders comprised of the
    protein tubulin
       a tubulin and b tubulin
   Outer diameter of 25 nm
   Much more rigid than actin
   Long and straight and usually have one
    end attached to a single microtubule
    organizing center
   Structures are dynamic with a fast-
    growing “+” end.
   Play a key role in structure of cilia and
    flagella where they provide rigidity and
    assist in generating motion.
   Play a key role in cell division in forming
    the mitotic spindle.
Intermediate filaments
   Ropelike fibers with a
    diameter around 10 nm
   Large and
    heterogeneous family
    of proteins
   Form a meshwork
    across the cytoplasm
    providing mechanical
     Cell classification
   Tissue of origin
       Lung, Liver, Kidney, Muscle, Blood (lymphocyte)
   History
       Primary cells
       Cell line
   Adhesion to surfaces
       Anchorage-dependent
       Anchorage-independent
           suspension
   Growth behavior
       Normal
       Transformed
   Morphology
       Cuboidal, spherical,
       Extended, compact
  Adhesion to surfaces
     Epithelial cells                      Macrophage cells
  Anchorage - dependent                  Anchorage - independent

Limited by available surface area    Not limited by available surface area

Behave like their tissue of origin   Behave like a different, amorphous cell
Anchorage dependent epithelial

macropages / monocytes

Epithelial cells attached to microcarriers (2D
surface on a 3D material)
Growth behavior
   Normal cells                   Transformed cells
       Anchorage-                     Non-anchorage
        dependent                       dependent (can grow in
       Mortal (reproduce for           suspension)
        a limited # of times)
                                       Immortal
       Form monolayers
        (contact inhibition)           No contact inhibition
       Require serum                   (can grow in layers)
        (growth factors)               May not need serum
       Highly differentiated          Lose differentiation
        Immortalized cell lines
   Mouse-mouse hybridoma cells (the
    fusion partners of the lymphocyte
    and the tumor cell)
   Chinese hamster ovary (CHO) cells
   Baby hamster kidney (BHK) cells
   HeLa cells
        named after a woman (Henrietta
         Lach) who had a cancerous tumor
         removed in the 1950's. Her cells
         continue to reproduce in cultures and
         are a key source for medical study.

                                                              CHO cells
Manipulation of cultured cells
   As cells generally continue to divide in culture, they generally grow to fill
    the available area or volume. This can generate several issues:
        Nutrient depletion in the growth media
        Accumulation of apoptotic/necrotic (dead) cells.
        Cell-to-cell contact can stimulate cell cycle arrest, causing cells to stop dividing
         known as contact inhibition.
        Cell-to-cell contact can stimulate promiscuous and unwanted cellular

   These issues can be dealt with using tissue culture methods that rely on
    sterile technique. These methods aim to avoid contamination with bacteria
    or yeast that will compete with mammalian cells for nutrients and/or cause
    cell infection and cell death. Manipulations are typically carried out in a
    biosafety hood or laminar flow cabinet to exclude contaminating micro-
    organisms. Antibiotics can also be added to the growth media.

   Amongst the common manipulations carried out on culture cells are media
    changes, passaging cells, and transfecting cells.
     Passaging cells
   Passaging or splitting cells involves transferring
    a small number of cells into a new vessel.
   Cells can be cultured for a longer time if they
    are split regularly, as it avoids the senescence
    associated with prolonged high cell density.
   Suspension cultures are easily passaged with a
    small amount of culture containing a few cells
    diluted in a larger volume of fresh media.
   For adherent cultures, cells first need to be
    detached; this is commonly done with a
    mixture of trypsin-EDTA, however other
    enzyme mixes are now available for this
    purpose. A small number of detached cells can
    then be used to seed a new culture.
     Developing a cell line
1)    Clone product gene cDNA
2)    Subclone into a suitable mammalian expression vector w/ a
      selectable marker
3)    Transfect host cells with plasmid
4)    Expose cells to selection reagent (24 - 48hrs)
5)    Pick 10s to 1000s of clones / colonies of surviving cells
6)    Screen for productivity
7)    Identify clones for further analysis
8)    Introduce cells to suspension culture conditions
9)    Final screen of productivity
10)   Cell storage and banking (LN2)
    Requirements of host cells
    Support high-level product expression
        10 – 100 pg / cell-day
    Stable production (many months)
    Can be scaled up (100 – 10,000 L) and grown in
    Can reach high cell densities (> 5×106 – 108 cells / mL)
    Provides appropriate post-translational processing of
        Folding, glycosylation, packaging, secretion
    Can be characterized to assure freedom from adventitious
     agents (viruses, micoplasms)
    Common host cells
   Chinese Hamster Ovary (CHO)
   Baby Hamster Kidney (BHK)
   Lymphoma cell lines (NSO, SP2, YB2/0)
   Fibroblast cell lines (3T3)
   MDCK
   HeLA

                                           CHO cells
    Methods of immortalization
   SV-40 transformation
       Simian vacuolating virus 40 or Simian
        virus 40, a polyomavirus that is found
        in both monkeys and humans.
       SV40 is a small DNA virus that has the
        potential to cause tumors, but most
        often persists as a latent infection.
   SV-40, causes cultured fibroblasts from
    mouse, rat, and hamster to partially or
    entirely lose their growth requirements
    for serum, anchorage, and insulin like
    growth factors, and alters the
    organization of the actin cytoskeleton
    of the cell
   Transformation by SV40 virus induces
    the re-expression of characteristics
    found in fetal epidermis and
    monostratified epithelia

Negative impacts of cell
immortalization process

   Altered metabolism
   Altered cell signaling
   Increased rate of DNA mutation
       Lowered rate of repair mechanisms
   Decreased product formation (potentially)
       Selection pressure leads to increased presence of
        fast growing cells over time
   Changed growth habit
Cell performance analysis
   Cell number and viability (%)
       Membrane integrity
           Trypan blue exclusion
               An intact membrane keeps the trypan blue dye out of (excludes) of the cell
           Lactate dehydrogenase (LDH) release
               Enzymatic quantification
           Vital fluorescent dyes
               Converted inside the cell to a different color
   Cell function
       Cell metabolism and product formation
           Nutrient uptake
           Protein secretion
       Enzyme activity or inhibition
       Calcium uptake
       Electrical response
       Proliferation (growth rate)
    Hematocytometer counts

   Add a vital dye (trypan blue)
       Dead cells stain blue
       Living cells appear white
   Place cells on slide under a
    cover slip
   Count cells in 5 large boxes
     “Typical” growth rate for animal cells
   Replication measurement (growth rate)                          First order growth model
          Count # of cells before and after some
           incubation time
              Cannot be done reliably with anchorage
               dependent cells
              Depends on:
                                                                            mX
                       Batch vs. continuous flow feeding scheme
                       Anchorage vs. suspended cells

     X = 105 – 5x106 cells / mL
     Mcell ~ 2x10-10 g / cell                                           dt
     X = 0.02 – 1 g/L

     m= ?
     Doubling time = ~24 hours
     td = 24 hrs ----- m = ?                                       X = cells / mL
     m = 1/t ln(X/Xo)
                                                                   N = total number of cells
     m = 1/td ln(2)

     m = 0.069 hr-1
    Functional assays
   Metabolism
       MTT
           Most common functional assay.
           Measures the conversion of dye by mitochondrial succinate
       ATP bioluminescence
           Bioluminescent detection of ATP using the firefly luciferase enzyme
           ATP has been used as a tool to assess the functional integrity of living cells
            because all cells require ATP to remain alive.
           Cell injury and death result in the rapid decrease in cytoplasmic ATP.

   Protein production
           Cytokine release
               Quantified by immuno assay (ELISA)
                              Viability curve of cells exposed to
                              a soap (surfactant that disrupts cell membranes)

Relative Metabolic Activity


                                          Cells Alive              Cells Dead


                                  0.001        0.01         0.1          1      10
                                                 Triton concentration [mM]
Back to challenge of the day

   The new supplement is designed to
    increase the strength at which cells attach
    to surfaces.
   Your product is produced by anchorage
    dependent cells that are grown on the
    surface of microcarriers.
   Will the product have an impact on your
    rate of production?
Key points
   Animal cells used to generate proteins and
    viruses for pharmaceuticals
       These products differ from those produced by
   Cell environment must be carefully controlled
       Anchorage vs. non-anchorage cells
       Membrane interactions with surfaces
   Metabolic needs provided by culture media
       Serum adds growth factors and other low
        concentration compounds

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