CELL CULTURE
You are given a suspension of cells at a concentration of 5 × 10⁵ cells/ml. You are asked to plate the cells for single-cell cloning using a 24-well plate, where each well contains 500 μl of culture media and should ideally contain 1 cell.
How many times should you dilute the original cell suspension to achieve this?
先要知道final是5×10⁵
initial就是说明一个well里有500ul=0.5ml=1 cells
那1ml就是2cells
所以initial是2cells/ul
dilution factor套公式是final/initial=250k
怎么算dilution factor可以用either
250k=100*100*25 或 100*50*50
这时就是用总额1ml来算的话，应该是
10ul of 5×10⁵ + 990ul Media = 100x
10ul of 5×10³ + 990ul Media = 10000x
40ul of 5×10¹ + 960ul Media
其实也就是
5×10⁵ = 500k
5×10³=5k
5×10¹=50
500k÷5k÷50=2cells/ml
volume是算DF得出来的
10ul+990ul=100x
1000/10 DF
40+960/40=25x
20+980/20=50x
b4 subculture
acquisition, isolation, disaggregation, culture after seeding
human use JKEUPM
animal use IACUC
mouse embryo:
undifferentiated mesenchymal cells
full term 19-21d
organ form at 9d - cells w less tissue
13-14d w/o brain, heart
complete form at 18d
chick embryo:
larger than mouse
Cell disaggregation:
Primary explant
tissue fragment on slide + nutrient
wait cells to migrate
✅small tissue
❎slow, poor adhesiveness, not for low migratory
Enzymatic
common
protease cleave CAM
mammal: trypsin, pancreatin, hyaluronidase
nonmammal: trypzean, trypLE, pronase, accutase, papain
trypsin + EDTA as chelating agent - capture calcium away from cadherin
cruder, more effective
purer, less toxic
trypsin inhibitor use for serum-free medium
✅no problems of migration, higher no cells, shorter time
❎select cells resistant to protease & mechanical stress
Warm trypsin
stir 37C every 30min
✅large tissue in short time
❎adult tissue (fibrous CT), mechanical agitation
Cold trypsin
4C for 6-18h
✅less toxic, high efficiency, more differ cell types, more viable cells, no stir & incubation
Mechanical
force
✅reduce proteolytic damage, soft tissue, less time
❎may cause mechanical damage, lower viability than enzymatic, require more tissue
Methods
gentle = spillage & sieving
spillage: slice/scrape
sieving: press - plunger
syringing
pipetting
nonviable cells remove at 1st change of medium, gradually diluted out when cell proliferate
viable cells at interface between medium & Ficoll/metrizoate
Subculture
cell density = cells/cm2
occupy substrate
cell concentration exceed medium capacity
growth reduced
importance: hetero to homo cell line
cell line = presence of cell lineage
cell strain = clone cell lineage w specific properties
passage no = subculture
gen no = doubling
1. 2 flask
original passage no 2
original gen no 2
new passage no 3
new gen no 3 by +1 doubling
2. 4 flask
original passage no 2
original gen no 2
new passage no 3
new gen no 4 by +2 doubling
3. 8 flask
original passage no 3
original gen no 3
new passage no 4
new gen no 3+3 doubling
计算机算100%÷8
然后按x2 算x2达100%的次数 加在gen no
finite cell 20-80 gen, determine age with gen no
continuous cell determine age with passage no
Periodic medium change:
drop of pH - falls 0.4 pH per day need to be fed within 24-48h
cell concentration - high concentration exhaust medium faster
cell type - transformed cells deteriorate rapid at high density; normal cells stop divide, block in G1, deteriorate very little
morphology - unhealthy sign granularity, cytoplasmic vacuolation, detachment
Subculture of adherent:
alternative protease - pronase most effective but harmful, dispase & collagenase less toxic
density of culture - normal cells reach confluency, transformed shortly after confluency as deteriorate after 2 doublings, epithelial b4 confluency as difficult to trypsinize
exhaustion of medium - pH fall, cell density increase
time since last subculture - correct seeding density & subculture interval done by perform growth curve
routine passage leads to the repetition of a standard growth cycle→controls the seeding concentration, the subculture interval, the duration of experiment, and the appropriate times for sampling
Finite cell lines – reduce the cell concentration at subculture by 2-, 4-, 8-, or 16-fold to make the calculation of the number of population doubling easier
Continuous cell lines - subculture at 1x10⁴ and 5x10⁴ cells/ml; fragile cultures (endothelium and some early-passage epithelia) at 1x10⁵ cells/ml
a new culture always start with high seeding concentration, gradually reduce
subculture at mid of log & plateau
Subculture of suspension:
no trypsin, quicker, less traumatic, easy scale up, more cells w/o increase surface area
no replace feed
Cell characterization
after enrichment, characterize
validate/identify cell line & determine cell purity
after transformation, characterize
Importance:
validate identity from primary culture
validate identity of continuous cell lines after prolonged culture or transformation
validate suspected cross-contamination after prolonged culture or transformation
Consequence of prolong culture:
cross contamination - low level cannot be detect by STR or SNP
mislabeling - misidentify
genetic instability - microsatellite instability cause loss of heterozygosity - loss or gain of alleles by STR
microbial contamination - mycoplasma no DNA change but alter phenotype
culture heterogeneity
Key requirement:
validation testing
safety
culture behavior
functionality
abnormality
usage
Key terms:
misidentify = wrong donor
misclassified = correct donor wrong tissue - phenotype changes not genotype
authentication = confirm identity - prevent misidentify but not misclassified - must use genotype method
provenance = detailed records provide info on origin, handling, validation, behavior
misidentified cell line:
lower risk obtain from primary source, higher risk from secondary source
ICLAC 486 case w/o known authentic material
main cause of irreproducible research
cross-contamination as primary concern
cell line authentication:
to uniquely distinguish cell lines derived from individual donor
to monitor stability of general cell characteristics
Methods:
genotype-based - determine hereditary characteristics
STR:
detect STR loci by multiplex PCR
analyzed by capillary electrophoresis
then CLASTR STR search
determine identity of donor based on DNA
time consuming & costly - usually outsourced to provider
data interpretation = identity scores
same donor >80% match
differ <50%
further analysis if 55-80%
limitation -
cannot distinguish cell lines or types from same donor - may display differences
cytogenetic analysis:
sp & sex determined by karyotyping - may be sufficient to confirm intersp cross contamination
transformation status based on chromosome no
variety of chromosome banding & molecular cytogenetic techniques (M-FISH, spectral karyotyping) identify marker chromosome w specific ds or other individual cell line
seq analysis:
can compare to available online database
indicate mutations/genotype based info - somatic mutation during cancer development
epigenetic analysis:
epigenetic status for lineage development & differentiation
influenced by sp & culture conditions
assessment of DNA methylation
phenotype-based - physical appearance
phenotype based characterization:
assess cell morphology
behavior
exp of markers specific to type, lineage, tissue or ds
transcriptome
tissue or lineage markers
myosin for muscle, melanin for melanocyte
enzymatic markers
limitation - not suit for authentication
in vitro transformation
changes in differentiation status differ in loss or gain of markers
culture condition 2D/3D determine phenotype
morphological assessment:
basic & direct
quick, easy, low cost - only phase-contrast microscope
identify cell type based on variation of morphology - epithelial/fibroblastic
routine observation
detect possible cross contamination
limitation -
distinguish cross contamination w distinct morphology
require high exp in handling
not suit to characterize sp, lineage, transformation status
require add method to confirm type or origin
morphology influenced by density, stage of growth, adhesion to substrate or medium composition
interpretation subjective
gene/protein exp analysis:
specific mRNA/protein
gene exp - RT PCR, gene microarray
protein exp - immunocytochemistry, flow cytometry, Western blotting, ELISA, Ab microarray - determine cell/tissue fx
markers detected by binding of Ab to specific Ag
limitation -
depends on known & valid markers & availability of tools Ab
influenced by culture conditions & phenotype
microarray may require specific requirements & could be costly
Cell separation
physical: density gradient centrifugation, unit gravity sedimentation, centrifugal elutriation
antibody-based: magnetic sorting, FACS
multi step more than 2 density - low resolution, unable to isolate cell with close density
continuous isolate 2 cell type with close density
each well differ layer
density marker beads confirm cells at correct density
Cell cloning
primary culture to subculture into cell line, pick one cell to clone to cell strain
easy for continuous cell line as unlimited lifespan
difficult for finite cell as senescence
dilution cloning easy for attached cells in petri dish, multiwell plates or flasks; for suspension, seeding into agar gel/Methocel
consideration:
trypsin, feeding, non uniform distribution as incorrect seeding, plating efficiency
final concentration: 10 colonies/ml
plating efficiency: 0.5%
how many cells/ml should be seeded = 2000
as 2000 * 0.5% = 10 colonies/ml
final concentration: 50 colonies/ml
plating efficiency: 0.1%
50k cells should be seeded
abcd答案直接乘于%就找到了
ways to improve plating:
capillary tube, microdrop technique, simple dilution
conditions improve clonal growth:
feeder layers
feeder layers
medium
serum - fetal bovine
hormones - insulin, dexamethasone
conditioning - used medium
intermediary metabolites - oxoacid ( pyruvate, a-oxoglutarate) & nucleoside
CO2 - 2%
treatment of substrate - polylysine, fibronectin
trypsin - pure trypsin at 4C
Suspension cloning:
agar - reduced sulfate polysaccharide with lower temp 4C, can be manipulated more at 37C
Methocel - sol not gel, higher viscosity when warm, sediment slowly - underlay prevent attach at base - colonies at interface between agar & methocel
Isolation of clones:
monolayer cells into multiwell, trypsinize, lay flat
monolayer cells in petri dish - remove medium, place ceramic ring around colony isolated as physical barrier
Cell differentiation
in lab, cells more prone to proliferation
differentiation = exp of phenotype to perform actual fx
terminal differ = final stage of differ not progress beyond
dedifferentiation = revert back to non differ
differentiation = constitutive - RBC from HPC, adaptive - monocyte to macrophage when pro inflammatory stimuli
dedifferentiation = adaptive - phenotype regain - wound, selective - precursor w greater proliferative -liver -macrophage to monocyte
stage of differentiation:
constantly renewing tissue
not turnover rapidly - regain density, differentiation reinduced
differentiation = cell division reduced
proliferation incompatible with differentiation
tissue markers:
cell surface Ag
intermediate filament proteins
regulatory control
differentiation markers:
enzymes - constitutive level, induced level, isoenzyme polymorphisms
differentiated products & fx
Induction of differentiation:
c-c interaction
- homotypic CAM cadherin gap junction, diffusible homocrine factors
- heterotypic +ve acting KGF produced by dermal fibroblasts & induces epithelium, -ve acting PDGF & FGF-2 promote grow & self renewal of O-2A progenitor cell but inhibit differentiation, paracrine factors, ECM modifications
- in vitro: high density (histotypic), recombinant culture (organotypic), soluble paracrine factors (IL-6, KGF)
systemic factor - physio inducer hormone, vitamin, inorganic calcium; non physio inducer DMSO, HMBA, N-methyl acetamide, benzodiazepine, cytotoxic drug
- in vitro: medium additives planar-polar compounds, signal transduction & chromatin modifiers (PMA & valproic acid)
c-m - ECM coat (glycoprotein & proteoglycan) fibronectin, laminin, collagen for epithelial & endothelial, synthetic macromolecule for neurite extension
- in vitro: matrix products, collagen gel, matrigel, ECM
dynamic stress - tensile for skeletal, periodic for cardiac, pulsatile flow for endothelium, compressive for bone & cartilage
- in vitro: tensile & compressive force transducer, pulsatile pump
polarity & cell shape
- hepatocyte grow on collagen gel, gel shrink, flat cells to cuboidal/columnar, develop polarity, asymmetry, nucleus @ bottom, Golgi @ apical
- thyroid epithelium w filter well establish polarity, basal receptor TSH secrete triiodothyronine, apical release thyroglobulin
- membrane trafficking, secretion & receptor presentation & recycling, position of nucleus
- in vitro: filter well inserts, matrices & scaffolds, collagen gel contraction
O2 tension
- exp of fully keratinized squamous differentiation in skin requires positioning of epidermal cells in an organotypic construct at air-liquid interface
- location of alveolar type 2 cells at air/liquid interface for optimal type 2 differentiation
- tracheal epithelium become mucus secreting only at air-liquid interface
- air-liquid enhance gas exchange & O2 uptake w/o partial pressure & free radical toxic
- thin film mimic physiochemical, +D-PBSA on apical surface = complete medium
- in vitro: distance from surface of medium, O2 concentration in gas, O2 carrier
Cell Transformation
change of phenotype - associated with DNA mutation, genetic instability
finite to ∞
genetic instability = continuous cell line
heteroploid - genetic diversity
Immoratalization:
inactivation of a no of cell cycle regulatory genes
control of senescence - delete/mutate senescence gene, telomerase activated, synthesize telomere; viral gene (T gene, adenovirus E1a, HPV E6 & 7, EBV) inhibit Rb, p53, p21, p16
Aberrant growth control to higher saturation densities:
loss of contact inhibition - transformation foci grow on top one another
reduced density limitation - increase labeling index with 3Hthymidine
anchorage independence - loss of cell–cell recognition and reduced cell–cell adhesion leads to a disorganized growth pattern and the loss of contact inhibition of cell motility and density limitation
lower serum dependence - transformed cells produce own food
controlled by oncogenes - modify receptor/signal transduction gene
Malignancy:
invasiveness - tumor spheroid completely replace chick heart; in vitro filter well, cells penetrate ECM (matrigel)
angiogenesis - chick chorioallantoic membrane & rabbit corneal assay inhibit; in vitro seed endothelial on bead embed in ECM
Culture of Specific Cell Types
Epithelial = parenchyma
most cancer derivative
perform most fx of body
contaminated w fibroblast (weed)
prevent overgrowth of unwanted cells
manipulate nutritional requirement only support epithelial
serum = best food for fibroblast & inhibit epithelial proliferation
use selective, serum-free media
if direct isolate from donor, perform w collagenase, dispase, trypsin at 4C disperse stromal cell/fibroblast but leave epithelial in small clusters
Mesenchymal cells = stroma/supp tissue = structural, vascular, lining
fibroblast survive most mechanical & enzymatic - culture in simple media - synthesize type 1 collagen for skin
adipocyte difficult to prepare
smooth muscle cell isolate from vascular tissue & coculture w vascular endothelium
osteoblast + EDTA + collagenase + antiserum against collagen 1
Tumor cells
fail to grow in vitro as challenges:
differ nutritional requirement
attempts to remove stroma
dilution may dilute out autocrine/homocrine GF or
reduce no w stem cell properties
paracrine interaction w stroma
contaminants: fibroblast, endothelial cells, normal equivalents of neoplastic cells
Selective culture of tumor cells:
selective media - HITES w hydrocortisone, insulin, transferrin, estradiol, selenium for small cell lung cancer - antiserum against collagen 1
confluent feeder layers - prevent fibroblast & normal equivalent outgrow - tumor cell no contact inhibition attach to feeder, normal cell die slowly
suspension cloning - cancer anchorage independent - cloning-forming efficiency often <0.1%, not easy to propagate cells isolated from colonies
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