Final Cell Culture

Cell Counting = process of determining cells no 算细胞 importance, method, which method recommended

• importance of cell counting
• ensure accurate seeding densities 播种密度
• monitors growth rates 成长
• evaluates cytotoxicity or proliferation 细胞毒性与增殖
• standardize experimental conditions 
• methods
• manual counting
• hemacytometer slide 
• requires a minimum of 1x10^6 cells/mL
• use trypan blue for viability test 
• viable cells not stained 
• cheap
• slow
• prone to error
• low sensitivity 
• electronic counting 
• original resistance-based counter
• based on the change in current generated when a cell passes through a narrow orifice 孔
• image analysis software 
• scan stained & unstained cells in a special counting chamber & view 
• continuous monitoring of living cultures 
• work by counting cells or determine residual available growth area 
• rapid & low inherent 固有 error - more accurate 
• needs single cell suspension 
• flow cytometry 
• count by light scatter, fixed or unfixed cell population or by DNA fluorescent stain
• analyze a single cell stream for cell concentration 
• many parameters can be measured simultaneously 同时 - cell size, cell lineage, DNA content, viability (Propidium I), apoptosis (annexin V)
• needs single cell suspension 
• technically more complex 
• growth curve 
• for design experiment - know when to subculture 
• population doubling time & cell cycle time determine cell growth 
• cell most consistent & ideal for sampling @ log
• cell has altered characteristics @ plateau
• secrete more ECM - more difficult to disaggregate 分解
• saturation density 
• density of cells at which the plateau phase is reached 
• transformed cells reach higher cell density @ plateau 
• higher growth fraction & loss of density limitation 
• cell cycle time 
• to determine length of cell cycle & its constituent phase 
• determined by measuring DNA synthesis 
• use bromodeoxyuridine (BrdU) = analogue of nucleoside
• BrdU incorporated into replicating DNA, replace thymidine
• DNA-bound BrdU labelled with Ab 
• incorporation of BrdU is detected at intervals after labeling by immunofluorescence microscopy/flow cytometry 
• for flow cytometry, cells stained with propidium iodide & analyzed for BrdU incorporation vs DNA content 
• cell migration 
• detailed analysis of time-lapse video sequence
• image analysis of tracks made by the cell's phagocytosis in dishes coated with colloidal gold
• movement of cells thro porous membrane - chemotaxis assay in Boyden chamber/invasion assay in filter well 
• data interpretation 
• total cells, live cells, dead cells 
• doubling time 
• population growth curve 
• impact of treatment 
• tips for accurate counting 
• mix cells well b4 sampling
• avoid bubbles in slide
• count consistent squares
• perform in triplicates 三份
• limitations
• subjectivity 
• debris
• over or under staining 
• calibration for automated counters

Cytotoxicity = quality of being toxic to cells

• cytotoxicity testing = in vitro assay of cell growth, survival, migration, death 
• development of pharmaceuticals, cosmetics, food additives
• humane - reduce animal sacrifice
• economic - cell lines are cheaper
• end point measurement 
• cannot predict systemic & physiological effects 
• initial screening of compounds 
• microtitration assay 
• allow screening of large no of samples
• save time & cost 
• set up MTT plate 
• incubate for 2 population doubling time 
• + drug/toxin to cells when cells in exponential growth @ log
• remove drug & allow cells to recover in growth medium 
• after 2-3 PDT, remove medium, replace with MTT 
• after 3-4h, read on plate reader
• use absorbance plot inhibition curve
• calculate IC50 
• nature of assay
• viability
• survival
• metabolic changes
• genotoxicity & transformation
• irritancy 
• parameter in survival assay*
• concentration of agent 
• invariant 不变 agent concentrations
• duration of exposure
• time of exposure
• cell density during exposure
• medium constituents 
• solvents 
• survival curve 

• IC50
• graph determine cytotoxicity activity of drug/cytotoxin  
• % control plot against drug concentration
• result in sigmoidal curve 
• IC50 = concentration where 50% inhibition occurs
• lower IC50 higher potency 效力
• based on graph below, IC50 value @ x-axis = ?
• 50% of cell population will die at the ? concentration of drug

• Survival*
• 3D spheroids 
• simple 3D aggregates typically generated from single cell types
• use for disease modelling 
• pros: easy to generate, cost effective
• cons: difficult to maintain long term 
• organoids 
• complex organized 3D structures that recapitulate概括/similar to original organ 
• use for disease modelling, regenerative medicine, drug discovery
• pros: multiple cells in culture, more closely mimic tissue 
• cons: challenging to generate  

Cryopreservation = cell freezing 

• reason for cell freezing 
• contamination by microbe 
• cross contamination 
• misidentification due to careless handling
• need for distribution to other users
• genotypic drift due to genetic instability 
• phenotypic instability due to selection & dedifferentiation
• principle - minimize intracellular ice crystal formation & reduce cryogenic damage 
• slow freezing, rapid thawing
• freeze cells slowly 
• use hydrophilic cryoprotectant 
• DMSO - sequester 隔离 water 
• store at lowest possible temp
• thawing rapidly
• minimize ice crystal growth & generation of solute gradients formed as residual intracellular ice melts 
• cell concentration
• survive freezing at high cell concentration
• when cryogenic damage - cause cells leaky
• reduce viability on thawing 
• allow sufficient dilution of cryoprotectant at reseeding after thawing 
• no need centrifuge 
• freezing medium 
• serum
• increase concentration to 40%, 50% or 100% 
• media 
• cryoprotectant*
• DMSO penetrate cell better
• 5-15% concentration
• may be toxic in hematopoietic cells 
• induce cells to differentiate after thawing 
• glycerol 
• hematopoietic cells
• low risk to induce differentiation after thawing 

• why cooling rate of frozen cells is not linear 
• cells survive best cooled at 1°C/min, cooling rate influenced by 
• ambient temp 环境温度
• cooling rate is proportional to the difference in temperature between ampoules & ambient air 
• @ -70°C, cells cool rapidly to around -50°C 
• rate falls off significantly after that
• can leave ampoules @ -70°C overnight then transfer to liquid nitrogen 
• insulation 隔热 
• plastic ampoule - safe & convenient
• made of polypropylene 
• inexperienced user avoid glass ampoule 
• risk of explosion when thawed
• if glass used, make sure perfectly sealed & use vapor phase cooling 
• specific heat and volume of ampoule contents
• higher specific heat capacity, slower cooling rate 
• larger volume of ampoule content, slower cooling rate 
• latent heat absorption during freezing 
• slower cooling rate 
• Vitrification*
• ways to control cooling rate
• cotton wool & polystyrene foam boxes
• ampoule canes in tubular foam pipe insulation
• freezer neck plug 
• freezing container
• controlled-rate programmable freezer

3D Culture* types, adv disadv, compare 2D 3D

• organ culture - multiple type & lineage 
• whole organs/representative parts maintained as small fragments in culture 
• retain intrinsic distribution of participating cells 
• technique
• primary explant with gas-liquid interface 
• on semisolid gel substrate of agar/clotted plasma/raft of microporous filter, lens paper/rayon supported on stainless steel grid 
• allow gaseous & nutrient exchange
• retain spherical geometry 
• liquid level too shallow - induce surface tension
• liquid level too deep - inhibit gaseous exchange 
• filter well insert
• compare with cell culture 
• organ culture @ gas-liquid 
• cell culture immersed in solid substrates - outgrowth of cells - alter geometry
• organ culture maintain cellular associations 
• cell culture use cells disaggregated enzymatic/mechanical - intrinsic distribution destructed 
• organ culture no grow & differentiate - density limitation of cell proliferation, physical restriction of geometry 
• cell culture grows & differentiates to reach confluency 
• limitation
• no vascular system 
• experimental analysis depends largely on histological techniques 
• more difficult to prepare
• cannot be propagated - original donor 
• study behavior of integrated tissues rather than isolated cells 
• histotypic culture - one cell type & lineage 
• propagated cell lines are grown alone to high cell density in 3D matrix
• techniques
• gel & sponge technique 
• cell penetrate cellulose sponge facilitated by collagen coating 
• collagen gel
• Matrigel 
• spheroid 
• culture in gyrator shaker 
• cells reassociate into clusters 
• form tissue-like structures 
• rotating chamber system - Miniperm bioreactor 
• cells proliferate in small compartment cylinder with medium in large compartment - high cell concentration 
• cells & medium separated by semipermeable membrane 
• slow rotation ensure mixing 
• membrane can be changed w/o disturb cell
• hollow fiber 
• medium + 5% CO2 pump thro centers of capillaries 
• cells attach & grow at outside of capillary fibers 
• gas & nutrient permeable on fiber surface support cell growth 
• filter well inserts choice: 
• cell + matrix + filter 
• cell + matrix + filter + interactive cell @ underside filter 
• cell + interactive cell + matrix + filter
• cell + matrix + filter + interactive cell @ underside filter with matrix
• allows cells @ very high density to proliferate  
• organotypic culture - multiple type & lineage 
• recombined cells of different lineages in experimental determined ratios & spatial relationships to recreate a component of the organ under study 
• steps 
• determine cell types
• isolation
• culture until reach desired cell no
• according to experimental determined ratios & spatial relationships 
• culture cells with various techniques
• techniques
• filter well insert
• hollow tube/perfusion chamber 
• component 
• tissue cells - endothelial cells 
• interactive cells - dermal fibroblasts in skin, smooth muscle cells in blood vessels, glial cells in neural constructs 
• biodegradable scaffold to support structure - PGA, PLA, calcium phosphate 
• matrix - collagen to coat scaffold, to enhance cellular attachment 
• mechanical stress - tensile for muscle, compressive for cartilage & bone, pulsatile for blood vessels