Primary Culture

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Chapter 12

• Primary Culture

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Primary Culture

• Types of Primary Cell Culture
• Stage after isolation of the cells but before
  first subculture
• Four stages
• Acquisition of sample
• Isolation of the tissue
• Allow cells to migrate from fragments of tissue
  adhering to substrate
• Disaggregating the tissue mechanically
• Disaggregate enzymatically
• Untransformed cells appear to need attachment to
  flat surface to survive/proliferate with max
  efficiency
• Transformed cells generally proliferate in
  suspension
• Dissection/disaggregation
• Culture after seeding into the culture vessel

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Primary Culture

• Types of Primary Cell Culture
• Common conditions needed for most cell lines
• Fat and necrotic tissue best removed during
  dissection
• Chop tissue finely with sharp instruments to
  cause minimal damage
• Concentration of cells needs to be much higher
  than for subculture
• Surviving tissue from primary culture can be
  quite low
• Rich medium preferable to simple medium (Eagles
  MEM)
• Fetal bovine serum better than calf or horse
• Selective media may be needed for specific cell
  types
• Embryonic tissue
• Disaggregates readily
• Yields more viable cells
• Proliferates more rapidly than adult tissue

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Isolation of the Tissue

• Introduction
• Human or animal tissues
• Work must fit within ethical rules
• Human tissue in Level 2 containment
• Site prep
• Clean/sterilize with 70 ETOH
• Remove tissue aseptically
• Transfer to culture lab from a dissection area

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Isolation of the Tissue

• Mouse Embryo
• Convenient source of cells for undifferentiated
  fibroblastic cultures
• Chick Embryo
• Due to size, easier to dissect
• Provide mostly mesenchymal cells
• Proliferation analysis
• Substrate for viral propagation
• Organs larger
• Differentiated organ cells easier to separate
• May be subject to to animal rights legislation

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Isolation of the Tissue

• Human Biopsy Material
• Presents serious problems
• Necessary to obtain consent
• From hospital ethical committee
• Attending physician/surgeon
• Donor or patient
• Harvesting tissues
• Not always convenient to tissue culture labs
• Collection/storage system needed
• Receiving system needed for recording details of
  the source, tissue origin, pathology, etc
• Notification of tech on arrival
• Safety note
• Material handled under Level 2 conditions
• Screened for opportunistic pathogens
• Hepatitis
• HIV
• TB

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? Hep B Virus
HIV ?
Mycobacterium tuberculosis
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Primary Culture
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Primary Culture

• Techniques for disaggregation of tissue for
  primary culture (pg. 183)
• Mechanical techniques
• Involve dissection
• With or without maceration
• Works well with soft tissues
• Firmer tissues
• When size of viable yield not important
• Enzymatic disaggregation
• Gives better yield
• Only when more tissue available

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Primary Culture

• Primary Explant
• Original method to initiate tissue culture
• Tissue fragment
• Embedded in blood plasma/lymph
• Placed on coverslip
• Inverted over a concavity slide
• Clotted plasma holds tissue in place
• Examined with conventional microscope
• Useful for small amounts of tissue
• Skin biopsies-little risk of losing cells during
  other protocols
• Disadvantage
• Poor adhesiveness of some tissues

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Primary Culture

• Enzymatic disaggregation
• Cell-cell adhesion mediated by variety of
  glycopeptides
• Cell adhesion molecules or CAMs
• Some Ca dependent (cadherins)
• Sensitive to chelating agents
• EDTA/EGTA
• Integrins
• Bind to arginine-glycine-aspartic acid (RGD)
  domains
• Also have Ca domains
• Affected by Ca depletion
• Fibronectin and laminin
• Matrix and basement membrane components
• Protease sensitive
• Approach
• Use trypsin or trypsin/EDTA
• Add other proteases to improve disaggregation
• Delete trypsin if necessary to increase viability
• In general, increasing the purity of an enzyme
  will give better control and less toxicity with
  increased specificity but may result in less
  disaggregation activity

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Laminin
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Primary Culture

• Enzymatic disaggregation
• Using mechanical and enzymatic disaggregation
  avoids
• Selection by migration
• Yields higher number of cells that are more
  representative of the whole tissue
• However, selection based on protease-and
  mechanical stress-resistant cells

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Primary Culture

• Enzymatic disaggregation
• Embryonic tissue
• Disperses readily
• Gives higher yield of proliferating cells
• Increased difficulty as age increases due to
• Onset of differentiation
• Increase in fibrous connective tissue and
  extracellular matrix
• Reduction of undifferentiated cell pool
• Severity of disaggregation
• Trypsinization or agitation
• -Harms fragile tissues
• -Tumor cells

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Primary Culture

• Enzymatic disaggregation
• Trypsin grade
• Purer
• Less toxic
• More predictable
• Cruder
• More effective
• Other proteases may be present
• In practice
• Test small amount first for viable cell yield

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Primary Culture

• Warm Trypsin
• At 37oC
• Dissociated cells from trypsin bath
• Collect every half hour
• Removed trypsin by
• Centrifugation
• Neutralization with serum medium
• Usefulness
• Disaggregation of large amounts of tissue in
  short time
• Mouse embryos
• Chick embryos
• Not good for
• Fibrous connective tissue
• Mechanical agitation bad for sensitive cell types
• epithelium

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Primary Culture

• Trypsinization with Cold Pre-exposure
• Minimizes exposure to 37oC trypsin
• 4oC for 6-18 hrs
• Allows penetration of enzyme with little tryptic
  activity
• Gives
• Higher yield of viable cells
• Improved survival preserves more different cell
  types
• Convenient
• No stirring
• No centrifugation
• May be done overnight

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Primary Culture

• Other enzymatic procedures- Collagenase
• Effective for many tissue types
• Embryonic
• Adult
• Normal
• Malignant
• Tissues too fibrous
• Too sensitive to use trypsin
• Highly suitable for
• Human tumors
• Mouse kidney, human adult and fetal brain
• Liver, lung
• Particularly epithelium based tissues
• Requires LITTLE or NO mechanical agitation

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Primary Culture

• Summary
• Most vital stage
• Disaggregation of tissue
• Preparation of the tissue
• Tissue loss at this stage
• IRREVOCABLE
• MUST chose proper technique
• In general
• Trypsin more severe than Collagenase
• More effective in creating single-cell
  suspensions
• Collagenase
• Doesnt dissociate epithelial cells readily
• Used to separate epithelial cells from stromal
  cells

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Primary Culture

• One last thing.Record Keeping!
• Proper records VERY important
• Origin and derivation
• Species, sex, tissue from which it was derived
• Relevant pathology
• Disaggregation procedures used
• Obligatory for GLP
• Kept in
• Notebook or file
• Best to initiate computer database
• May become very important if cell line becomes
  important-repeatability!
• Authenticate origin of cell line
• Reference for DNA fingerprinting or profiling