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Week 7 + 8: Cellular Fate Processes, Week 9: Cell & Cell Cultures,…

- - - - Somatic (parent) cells are constantly dividing to produce new (daughter) cells
        
        To replace old, damaged, or dead cells
        
        To maintain body function when growing
      - Different than cell differentiation
        
        Where (stem) cells change phenotype
      - Process by which a parent cell divides into 2+ daughter cells
      - Cells control how & when they divide by communicating with one another
        
        Communicate using chemical signals
        
        Signals act like switches to communicate to cells when to start/stop dividing
        
        Its important for cells to stop dividing at the right time, otherwise it might lead to diseases such as cancer
    - - Process in which acell separates chromosomes into 2 identical sets, each with their own nuclei
        
        Daughter cells each have same # of chromosomes & DNA
      - Steps
        
        Two wheel shaped structures
    - - Creates sex cells
      - Only copies half of the genetic information
    - - Process which divides the cell contents into 2 cells
        
        Contain roughly equal shares of cellular componenets
  - - - Necrosis
        
        Unplanned (Unprogrammed) Cell Death
        
        Accidental cell death due to environmental perturbation or cell injury
        
        Leads to uncontrolled released of inflammatory content
        
        Leaked organelles & molecules from within the necrotic cell trigger an immune response
        
        Steps
        
        Swelling Stage
        
        Cellular injury (hypoxia or toxins) causes a decrease in ATP production
        
        Disables the ion balance within the cell
        
        Cell begins to swell
        
        Breakdown Stage
        
        Lysosomal enzymes (responsible for digestion of cell parts & macromolecules) leak out into cytoplasm
        
        Influx of lysosomal enzymes degrade other organelles, as well as nucleus
        
        Nucleus shrinks & dissolves
        
        Inflammatory Response
        
        Cell eventually loses its integrity as content leaks into cytoplasm
        
        Starts inflammatory response & bystander immune cells respond
        
        Examples
        
        Heart Attack (myocardial infarction)
        
        Necrosis occurs in cardiac cells
        
        Caused by decreased blood flow & oxygen
        
        Stroke
        
        Toxins
      - Apoptosis
        
        Planned (Programmed) Cell Death
        
        Programmed Process of autonomous cellular dismantling
        
        Avoids inflammatory reaction
        
        Controlled process, no inflammatory immune response
        
        No leakage of cellular components
        
        Steps
        
        Signalling Stage
        
        Tightly controlled structure & process
        
        Cell decides to die off due to intrinsic/extrinsic pathways
        
        e.g. receptors on cell receives signals from outside
        
        This signalling pathway creates a specific enzyme called "caspases"
        
        Condensation Stage
        
        Chromatin condensation causes phase change from heterogeneous & genetically active material to inert material
        
        This then causes the cell to fragment into apototic bodies
        
        Fragmentation Stage
        
        Capases activation causes nuclear fragmentation & digestion of nucleus & other organelles
        
        Phagocytosis
        
        Apoptotic bodies are phagocytosed by macrophages
        
        Activates under the following conditions
        
        Physiological conditions, when body wants to get rid of unwanted cells during embryogenesis
        
        In response to cell injury, such as DNA damage
        
        If cell cannot undergo apoptosis, it can shift to necrosis
    - - Similar to division
        
        dX/dt = aX
        
        a = rate of apoptosis
        
        X = cell number
        
        t = time
      - Notes
        
        Process is activated due to several triggering biochemical events
        
        Process is irreversible
        
        The rate of apoptosis is fast (order of hours) than that of DNA synthesis in cell division
      - If rate of apoptosis is governed by an effector (C):
        
        a = amax[C]/(Km + [C])
      - May not need to know for exam
  - - - Directed movement of a single/group of cells in response to chemical or mechanical signals
        
        Our body relies on cells being at the right place at the right time
      - Migration (or motility) plays an important role in both physiological & pathological processes
        
        Embryogenesis process & Organogenesis
        
        Wound healing & ECM remodelling
        
        Tumour formation
      - While few cells are considered immotile (e.g. osteocytes), most cells migrate in response to different stimuli
    - - Skin cells
        
        Keratinocyte stem cells differentiate & migrate to the outer most layer of epidermal layer
        
        Repeats continuously
      - White Blood Cells (WBCs)
        
        When your immune system finds a harmful substance, it releases cytokines at the site of infections
        
        Cytokines trigger positive chemotaxis (chemical gradient)
        
        Draws immune cells to site of injury
      - Torn tissue healing
        
        Body starts making new cells
        
        Guides them to move to site of injury to fill wound
      - Tumour formation
        
        Cancer cells tunnel through ECM & invade neighbouring tissues (metastasis)
        
        I.e. breast cancer cells moving to bone or brain
    - - Protrusion of Lamelliphodia
        
        Internal forces that induce the cell to polymerize F-actin
        
        Dynamic structural protein within cytoskeleton
        
        Induce the formation of the protrusion
      - Adhesion of the leading edge through integrin receptors
        
        Leading edge accumulates new actin protein to the protrusion edge
        
        Adhesion of the leading edge through integrin receptor
      - Movement of the Cell Body
        
        Contraction of the cell body in the direction of movement
      - Deadhesion of Trailing Edge
        
        Integrin receptors in the back of the cell decrease
        
        New adhesion molecules appear in the front
    - - Whole Populations
        
        dX/dx = J
        
        J = flux vector at the system boundary
        
        cells*distance/time
        
        X = cell number
        
        x = distance
        
        Cellular fluxes
        
        Chemotaxis
        
        Chemically prompted kinesis that causes cells to become motile or change their direction in response to chemical signals
        
        Chemokinesis
        
        Random, non-vectorial movement of cells
        
        -sigma(dX/dx) + xX(dA/dx)
        
        X = Cell number
        
        x = distance
        
        x = Chemotactic Coefficient
        
        distance^2/(time*concentration)
        
        A = Concentration of chemo attractant
      - Individual Cells
        
        Motile cells appear to undergo a random walk process that can also be described as a diffusive type process with motility characteristics measured on individual cells
        
        Described in terms of cell speed (s) & persistence time (p), which is the length of time that the cell moves without changing its direction significantly
        
        sigma = (1/2)(s^2)p
        
        sigma = random motility coefficient
        
        distance^2/time
  - - - Transdifferentiation
      - iPSC
    - - Continuous
        
        Uses cell stage as a continuous variable
      - Compartment
        
        Occurs as a series of discrete changes, with all cells at a particular stage (compartment) being homogeneous in nature
        
        I
        
        X1
        
        X2
        
        ...
        
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        Rate of Change between Compartments
        
        dXi/dt = k(i-1) X(i-1) - k_i X_i
        
        For Steady State (ss)
        
        X1 = I/k1
        
        X2 = I/k2
        
        k_i = transition rate of cells at stage Xi to stage Xi+1
        
        I = # of cells in the input flux
        
        X_i = # cells at ith stage of differentiation
        
        Uses compartments for each stage of differentiation
- - - - Compartment Model
        
        Uses compartments for each stage of differentiation
        
        Occurs as a series of discrete changes in cell phenotype, with all cells at a particular stage (compartment) being homogeneous in nature
        
        Rate of change: dXi/dt = k(i-1)X(i-1) - kiXi
        
        Steady-State (StSt) # of cells at any particular differentiation stage:
        
        X1 = I/k1
        
        X2 = k1X1/k2 = I/k2
        
        Overall time constant can be estimated as:
        
        See Week 8, Slide 4
        
        tau_diff = sum(1/ki)
      - Continuous Model
        
        Uses cell stage as a continuous variable
        
        Whole process proceeds at a fixed rate
        
        1st order ODE that relates differentiation as a linear process
        
        See Week 8, Slide 5
      - Neither approach use any molecular level info
        
        Use phenomenological cell population-type models
- - - - G0 Phase
        
        Cell Cycle Arrest
        
        Cells in resting state until signalled to divide
        
        Duplicates everything except chromosomes
      - Cell grows & DNA replicates
      - G2 Phase
        
        Cell growth, protein synthesis
        
        Checks DNA integrity & replication
        
        Apoptosis if there are problems in replication
      - S Phase
        
        Chromosomes (DNA) duplicated
        
        Cell is irreversibly committed to division
      - G1 Phase
        
        Cellular content duplication
        
        Cells grow, make protein & energy
        
        Where cell chooses whether or not to divide
    - - Replicated DNA & cytoplasmic contents separate
        
        Cell Divides
      - Spindle Fibres (special proteins) help equally divide chromosomes
        
        Cycle will not proceed until all chromosomes are firmly attached to 2 spindle fibres
      - 5 Steps
        
        Prophase
        
        Two wheel-shaped structures (centrioles) position at the opposite end of cells, forming poles
        
        Still Prophase
        
        Long proteins (mircrotubules) extend from centrioles in all possible directions, forming spindles
        
        Metaphase
        
        Some spindle fibres connect poles to kinetochores (proteins near centromere)
        
        Still Metaphase
        
        Chromosomes pull back & forth until aligned near equator
        
        Anaphase
        
        After checkpoint, the spindles pull chromosome toward each pole
- - - - Such as O2 & CO2 concentration
  - - - 1-log of cells
  - - - These techniques are required to
        
        Expand key cellular components
        
        i.e. Growing cells for autologous transplant
        
        Investigate determinants of cell fate
        
        i.e. Cel differentiation in embryogenesis or iPSC engineering
        
        Investigate Maturation of engineering products in-vitro
        
        i.e. Simulate tissue function to mimic in-vivo conditions
        
        Maintain/Transport mature constructs for clinical applications
        
        i.e. Transport cells from one place to another
- - - - Anchorage dependent cells
        
        Majority of cells in body
      - Growth of cell occurs attached to the surface of the flask
      - Cells require substrate for growth
        
        Promote cell-to-cell interractions
      - Most immobile cells grow in an anchorage-dependent manner
        
        (Except hematopoietic cells)
    - - Anchorage independent cells
      - Cells float in medium
        
        Can form cell aggregates
      - No substrate required for cell attachment
      - Require continuous agitation (stirring, mixing) for adequate gas exchange (e.g. oxygen)
    - - Scaffolds or templates
      - Interact in 3D environment, rather than 2D plates
- - - - But allows easy visual inspection under microscope
    - - To detach from surface they are adhered to
    - - May limit product yield
    - - Harvesting on small scale, microtechnology, cancer screening
  - - - But require daily cell counts & viability assessment to follow growth patterns
    - - Because they floating
    - - Allows easy scaling
    - - But require agitation for gaseous exchange
- - - - Simple, low cost, low maintenance
      - Compatible w/ existing functional testing
      - Unlimited supply of nutrients, oxygen
        
        Vs. in-vivo or 3D
    - - 2D dish can't mimic natural structures of tissues
      - Cell-cell & cell-ECM interaction not represented
      - 2D culture can lead to attend morphage & cell division
      - Allows for culturing of only one type of cell
  - - - More natural cell-cell & cell-ECM interaction
      - More accurate morphology in-vivo like cells
      - Improved cellular topology, gene expression, signalling & metabolism
    - - Cells attached to the scaffolds flatten
        
        Then spread like 2D adherent cell cultures
      - Topology & scale of scaffold can create some undesirable results
      - Scaffold material can impact adhesion, growth, & cell behaviour
      - Cell observation & extraction can be more challenging
- - - - Cells isolated directly from human/animal tissue enzymatically or mechanically
        
        Placed in tissue culture
        
        e.g. adherent or suspension
      - Advantages
        
        Can study the donor & not just the cell
        
        Risk factor (age, history, gender, race) can be considered in experimental medicine
        
        Opportunities for personalized medicine
      - Disadvantages
        
        Limited life span
        
        Possibility of viruses or bacteria
    - - Cells that have been continually passaged over long period of time
        
        Have acquired homogeneous genotypic, & phenotypic characteristics
      - 2 types
        
        Passaged Cells
        
        Divided cells during culture, which can be used for multiple generations
        
        Process of re-planting divided cells into new cultures is called passaging
        
        Not all cells can be passaged
        
        Human fibroblast & T-cells do well in culture, but cardiomyocytes die within a few hours of culture
        
        Finite cell lines sub-cultured for ~20-80 passages
        
        Slow growth (>24 hours)
        
        Immortal Cells
        
        If cell line continues for extended period of time
        
        Ability to proliferate through genetic mutations or artificial modification
        
        Divide rapidly (12-24 hours)
        
        Subcultured indefinitely
        
        Have altered growth properties
        
        Transformed cells can spontaneously arise by a few cells escaping deterioration
        
        e.g. naturally occurring cancerous cells
        
        Can occur due to
        
        Genetic instability
        
        Overexpression of telomerase
        
        Prevents chromosome degradation in DNA replication
        
        Mutations in the group of "senescence genes"
        
        Genes that are involved in the process of cellular senescence, a state of permanent cell cycle arrest
      - Advantages
        
        Can produce a large amount of homogeneous cell types
        
        Cell lines are convenient (easy to handle)
      - Disadvantages
        
        Less preferred for biological application
        
        Because cells have lost their true characteristics
        
        Serial Passaging
        
        Phenotypic/genotypic alterations
        
        Variation can be so large that they may no longer mimic in-vivo conditions
        
        Result in false data
    - - At about 5 weeks, a Primary culture becomes a cell line
        
        At about 12 weeks cell line becomes a continuous cell line, or dies
- - - - Contain human papilloma virus 18 (HPV18)
        
        Responsible for HPV-caused cancers
    - - Telomerase shortening doesn't happen in HeLa cells as is common in other cells related to natural aging & death
    - - However, these cell lines have a tendency to change over a period of time
    - - Telomerase
        
        Grow rapidly for cancer cells due to an active version of telomerase that prevents telomere shortening associated w/ the normal aging process
      - Chromosome Numbers
        
        Transfer of HPV18 into human cervical cells lead to HeLa genome
        
        Consisting of 76-80 chromosomes, rather than the 46 in a healthy human
      - Contamination
        
        HeLa cells are so prolific that they travelled through dust particles & invaded other cell cultures in labs
        
        Leading to mislabelling & world-wide pervasive cross-contamination
    - - Biological specimens were considered property of the physician, not the patient
      - Segregation was still legal in 1950
        
        So Henrietta didn't have access to many hospitals
      - Dr. Grey gave away the HeLa cell line to anyone who requested for free
        
        Could have been very lucrative to the Lacks family
        
        But HeLa cells have been used in Polio & COVID-19 vaccines
        
        And to study leukemia, AIDs, and cancer
      - In 2023, researchers published a detailed genomic analysis of HeLa cell line (DNA & RNA profile)
        
        Raised concerns of genetic privacy, and legal/ethical norms in genetic research
        
        Lacks family protested the publication since it disclosed important health data that could affect insurance, disability coverage, and unfair disclination
        
        After scientists determined it was possible to extract genetic info, they retracted the genetic profile and made it available on a controlled-access basis
- - - - Class I
        
        Protection to lab personnel
      - Class II
        
        Provide protection as well as an aseptic environment to cell/tissue cultures
        
        Also for hazardous materials
      - Class III
        
        Highest attainable level of protection to personnel & environment
        
        Required for work involving known human pathogens
    - - 70% is blown down on surface after passing through another HEPA filter
  - - - Temp, CO2, Humidity
        
        Temp = 37 +/- 0.2 C, Humidity Degree = 95%, CO2 = 5 +/- 0.5%
        
        CO2 for maintaining cell pH
- - - - Vol of Medium = (Cells Require) / (Cell Density) = (10^9 cells) / (10^6 cells/mL) = 1000 mL = 1L
        
        A typical bioreactor can hold ~10L of fluid, so we can produce
        
        number batches per bioreactor = 10L / 1L = 10 batches
        
        1L of cell medium
- - - - These 2 criteria have led to several design-related challenges
        
        Oxygenation
        
        Providing adequate flux of oxygen at physiological concentration & removal of CO2
        
        Provision/removal
        
        of cytokines, chemokines, & any other macromolectules
        
        Physiological perfusion rates & uniform flow distribution down to small dimensions
        
        ~100 um
- - - - e.g. Protein diffuses 1mm in 1 hr, but it would take 100 hrs to diffuse 10mm
        
        Thus, it is challenging to provide adequate mass transfer into a cell bed of physiological cell density
  - - - If nutrient delivery is slow compared to consumption
        
        Local nutrient concentrations drop to unacceptable levels
        
        Rate of change of mass of a particular nutrient or waste product can be determined with Consumption Rate:
        
        dm/dt = qXV
        
        V = Volume of vessel (mL)
        
        q = Specific nutrient consumption rate (umol/cell/hr)
        
        X = # of viable cells/unit volume (cells/mL)
      - If nutrient delivery is too high
        
        Local nutrient concentration can build up to inhibitory levels
  - - - Physiological Concentration
        
        5-30% of saturation relative to air
        
        ~0.2 mM
      - Specific uptake rate
        
        0.05-1 umol/10^6 cells/hr
    - - Physiological Concentration
        
        mM range
      - Specific uptake rate
        
        0.05-0.1 umol/10^6 cells/hr
    - - Physiological Concentration
        
        nM - uM range
      - Specific uptake rate
        
        0.01-1 nmol/10^6 cells/hr
    - - q in equation
      - Physiological concentration
        
        Negligible
      - Specific production rate
        
        0.01-0.2 umol/10^6 cells/hr
- - - - Waste Products
        
        CO2
        
        Lactic Acid
        
        Ammonia
      - Cytokines
        
        Inflammatory Cytokines
        
        IL-1, IL- 10, TNF-a
        
        Reactive Oxygen Species
      - Degenerative Enzymes
        
        Matrix Metalloproteinases (MMPs)
  - - - Lead to differences in the local concentration of solutes vs. the mid-line flow
        
        Flow at the boundary layer is inversely proportional to the thickness of its thickness
        
        Influences cell & tissue growth
        
        Induces or alters cellular migration
        
        Chemotaxis
        
        Directed movement of cells toward/away from chemical source
  - - - tau_r = V/F
        
        tau_r = residence time (min)
        
        V = Vessel Volume (bioreactor) (mL)
        
        F = Volumetric Flow-Rate
    - - But any non-uniformity in flow conditions can lead to distribution in residence time
        
        Can cause problems or lead to excessive variability in cell/tissue growth
- - - - Economical
      - Many sizes
    - - Requires constant media changes
        
        Makes it labour intensive & cost inefficient
  - - - Large surface area
    - - Must be treated as single unit w/ all cells in each layer seeded, cultured, & detached at the same time
        
        Multiple layers and weight make this hard
  - - - Rotation provides mixing
    - - Still require regular media change
      - Require extra step of rotating flasks
        
        Requires electricity
  - - - Provide high cell numbers & better aeration
        
        Due to mixing feature
    - - Shear stress caused by rotation can damage cells
- - - - Most common justification for ethical decisions in modern society
        
        e.g. taxes or mandatory seatbelts
      - Conflict
        
        Can arise when evaluating the benefits or when distributing them equally
        
        Benefit must not favour special groups or people
  - - - Universal good will
        
        Be kind, don't cheat, don't lie, etc.
        
        Conflict
        
        Can arise when following universal principals may cause harm
        
        e.g. Telling white lies is not acceptable, even if the truth causes harm
  - - - Individuals' rights are basic and others have no right to infringe on them
      - Conflict
        
        Challenging to determine when one person's rights infringe on anothers
        
        People occasionally claim self-serving "rights"
  - - - Developing virtuous qualities using thought
      - Conflict
        
        The definition of virtue is often vague & difficult to apply
- - - - Mill would approve of one life for five as the greatest good for the greatest number of people
    - - Would object to taking any life & let the train continue in both cases
- - - - Dr. David Golde & Dr. Shirley Quan recommended spleen removal
        
        Moore consented
        
        However, Dr.s took part of the spleen for research, which Moore did not consent to
        
        Doctors found Moore T-lymphocytes interesting b/c they overproduced certain lymphokines
        
        Dr. Golde instructed Moore to keep visiting him from 1976-1983
        
        During this time, he kept taking samples under the guise of necessary treatment
        
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