Medical Sciences - Week 5

How do you select the right cell line

• species

  • choose based on experiment needs

  • nonhuman/nonprimate cell lines have fewer biosaftey concerns

• function

  • lines that align with exp goal

• finite vs continuous

  • finite cell line: more accurate functions

  • continous cell line: easuer to clone & maintain

• normal vs transformed

  • transformed: faster & less serum

  • undergone genetic changes

• growth characteristics

  • speed, cloning ability, can it grow in suspension

how do you acquire cell line

• reputable suppliers (cell banks)

• culture from primary cells

*mycoplasma (bacteria) contamination before use

what factors can you control (adjust) in a cell line?

temperature, pH & gas levels (O₂ and CO₂).

what is media?

• contains nutrients & hormones

what is adherent culture?

• Cells grow on surfaces (like tissue culture-treated plastic)

• Most vertebrate (including mammalian) cells need a surface to grow on.

What is suspension culture?

Cells grow free-floating, often with magnetic stirrers

This is useful for large-scale production

what cell types do you use adherent culture vs suspension?

adherent: most cell types (primary)

suspension: cells that are nonadhesive

how do you passage adherent culture vs suspension?

adherent: periodic, but easy visual inspection

suspension: easier, but require daily cell counts & viability determination

how do you disassociate adherent culture vs suspension?

adherent: enzymatically (trypsin) or mechanically

suspensions: not require enzyme or mechanical

adherent culture vs suspension is limited by?

adherent: surface area

suspension: concentration of cells in medium, easier to scale up

how do you maintian adherent culture vs suspension?

adherent cells: require tissue culture-treated vessel

suspension: no tissues needed but agitation (shaking or stirring) for adequate gas exchange

what are adherent cells vs suspension cells used for ?

adherent: cytology, harvesting products

suspension: bulk production, batch harvesting

& many research applications

what are the types of media ?

basal: nutrients but no additional growth factors so needs serum

reduced-serum: enriched with nutrients so needs less serum

serum free media (SFM): doesn’t use animal serum, instead tailored w hormones and gf for specific cells

what is serum

liquid part of blood  that contains GF, hormones, nutrients & protiens

What are the advantages of serum free media?

• consistent performance

• easier purification & downstream processising

• percise evalutation of cellular function

• increased productivity

• better control over physiological response

• enhanced detection of cellular media

What are the disadvantages of serum free media?

• requirement for cell type-dpecific media formulations

• need higher degree of reagent purity

• slower growth

what temperature do you store cell culture

• refrigerated (2-8 deg celcius)

• warm beofre use

keep your cell culture protected form […]

• light

• light exposure degrades the essential vitamins that it needs to grow

when should u use the supplemented media within and to make sure what

2-4 weeks

reduce chance of contamination and impact of pH drift

what is the purpose of serum

• provides essential growth factors, hormones, lipids, and minerals for cell growth

• it also regulates cell membrane permeability and acts as a carrier for lipids, enzymes, and trace elements into cells.

what is FBS

• fetal bovine serum

• preferred over other animal sera (horse, rabbit, goat) due to:

  • Low gamma globulin content.

  • High growth factors.

  • Fewer complement proteins (immune system), reducing the risk of cell lysis.

what are the advantages of FBS

effctive growth w fewer applications

what are the challenges of FBS

• expensive

• it is hard to standardize (variability)

• contamination risk if purachse from bed sources

what are cell culture plastics treated with so cells can attach

• first : polystyrene (PS) - hydrophobic

• second: peptides + proteins - make more hydrophilic

• tissue treated: hydrophilic which allows extracellular matrix proteins to adhere

what are cell culutre vessels

• dishes

• flasks

• roller bottles & spinner flasks

• cell culture plastics

  • untreated

  • tissue treated

  • or treated with other materials

• chambers: adherent cells on a microscope slide, can be treated to improve attachment

• tubes: centrifuging, culturing or storing

what pH do mamalian vs insect cells grow at

• pH: Most mammalian cells grow at pH 7.4; insect cells prefer pH 6.2.

what does CO2 kept at with cultures?

Helps buffer the pH in cell cultures, usually maintained at 5-7%

most human/mammalian cells grow at […]°C; insect cells at […]°C; avian cells at […]°C.

mamalian 36–37°C; insect cells at 27°C; avian cells at 38.5°C.

what is the improtance of cell morphology

• regularly check shape and apperence to ensure healthy cells

• detect signs of contamination

what is the morphology of mamalian cells?

• Fibroblastic (fibroblast-like):

  • bipolar/multipolar & elongated

  • attached to substrate

• Epithelial-like:

  • Polygonal, growing in patches

  • attached to substrate

• Lymphoblast-like:

  • Spherical

  • usually grows in suspension without attaching to a surface.

some specialized cells have distinct morphologies based on their […] such as […] cells.

• specialized cells have distinct morphologies based on their functions, such as neuronal cells.

neuronal cells is divided into two basic types

• Type I: Long axons for signal transmission over long distances

• Type II: No axons.

neuronal cells can be:

• Unipolar or pseudounipolar: Dendrite and axon come from the same process.

• Bipolar: Axon and single dendrite emerge from opposite ends of the cell body.

• Multipolar: Multiple dendrites with the cell body containing the nucleus.

what are 293 cells?

• derived from human embryonic kidney

• transformed with adenovirus type 5 DNA. -allowing them to become immortalized

• produce recombinant pro(plasma) tiens (

• Variants available for high-density suspension culture in serum-free media.

• Adherent cultures should be passaged before reaching full confluence, ideally in the log phase

what is subculturing or passaging?

removing old media and transfering cells from existing culture to fresh growth media

what is the purpose of subculturing

This process helps continue the propagation of a cell line

What are the growth phases of cell lines?

• Lag Phase: Initial slow growth after seeding.

• Log Phase: Cells proliferate rapidly and exponentially.

when do you subculture adherent cells vs suspensions

• Adherent Cultures: When cells cover the entire substrate with no space left for expansion.

• Suspension Cultures: When cell density becomes too high for the medium to support further growth.

what is the goal of subculturing

To maintain optimal cell density and stimulate further cell proliferation by splitting the culture and providing fresh medium.

for mamalian cells what phase you subculture at?

• Subculture during the log phase before reaching confluency.

what do normal cells vs transformed cells stop growing at for adherent cultures?

• Normal cells stop growing due to contact inhibition at confluency, and recovery is slower after reseeding.

• Transformed cells can continue to grow past confluency but deteriorate after about two doublings.

when are suspension cultures subcultures?

• Subculture during the log phase, before confluency.

what happens at confluency in suspension cultures?

• At confluency, cells in suspension clump together, making the medium turbid.

in mammalian cells when pH drops what does this indicate?

• pH drop indicates the buildup of lactic acid, which can be toxic to cells.

• Subculture if a rapid pH drop (more than 0.1–0.2 units) occurs alongside increased cell concentration.

what are the two ways to mechanically disassociate cells?

• shake off

  • gentle shaking

  • rigorus pipetting

• scapring

  • cell scraper

what are the 4 ways to enzymatically disassociate cells?

• trypsin (strongly adherent cell cultures)

• trypsin & collagenous (multiple layers like fibroblasts)

• dispase (epidermal cells)

• TrypLE dissociation enzyme (strongly adherent cells)

  • substitute for trypsin

what is the protocol for passaging adherent cells

1. sterile env

2. remove old medium (spent media)

3. wash cells (used balanced salt solution w out Mg&Ca to wash cells, add gently to not disrupt cells & gently rock)

4. discard wash solution (remove & discard wash solution )

5. add dissociation reagent (trypsin or TrypLW & gently rock)

6. incubate: incubate @room temp for 2 mins

7. observe attachment: check that at least 90% detachment

8. neutralize the reagent: add growth medium to neutralize the dissociation reagent. Pipette over the cell layer to mix

9. centriuge

10. resuspend cells & add growth media

11. count cells:Determine total cell count and viability using a hemocytometer or cell counter

12. seed new culture: diltue suspension & transfer to new vessels & incubate

Passaging suspension cells is […] than adherent cells since

easier

they’re already suspended in the medium, so no enzymes are needed to detach them

 Unlike adherent cells, suspension cultures aren’t […]

Instead, […] is done every 2–3 days to maintain growth.

fully replaced with fresh medium.

feeding

what are the two methods of passaging?

1. Dilution: Directly dilute the cells in the culture flask to keep expanding them.

2. Partial Removal: Take out a portion of the cells, then dilute the rest to the proper seeding density.

 Suspension cells usually have a […] after passaging compared to adherent cells

shorter lag period

What is cryopreservation?

Cell lines are vulnerable to genetic drift, senescence, contamination, and equipment failure. Cryopreservation ensures long-term storage of valuable cell lines.

when to freeze cells?

As soon as there is a surplus of cells from subculturing, freeze them as seed stock. Seed stocks should be protected and not used for routine lab work. Working stocks can be replenished from these frozen cells.

how do you do Cryopreservation

Cells should be stored in liquid nitrogen with a cryoprotective agent, such as DMSO (dimethylsulfoxide). This protects cells by lowering the freezing point and preventing ice crystal formation that can cause cell damage.

how do you handle DMSO

DMSO can allow harmful organic molecules into tissues, so handle it with care and dispose of it according to safety regulations.

what are three examples of 2D cells

ADHERENT

1. Primary cell culture: comes from human/animal tissue

2. Cell line: immortalized cell line, can continue to divide long term 

3. 2D co-culture: multiple types of cells growing 

what are examples of 3D cells?

SUSPENSION

1. Spheroid 

2.  Organoid: stem cells can develop as an organ would develop in the body 

3. 3D co-culture: multiple cells, organoid can grow into this  

[…] can cause transmissible disease

Biological Hazards: can cause transmissible disease

what are biosafety measures?

• Safety equipment (primary barriers [@ level of the hazard] ) 

  • Biosafety cabinet (BSCs), sealed containers (contains hazard) 

• Personal Protective Equipment: gloves, coats, glasses 

• Faculty Design & Construction (secondary barriers) 

  • Ventilation (filter pathogen from going out into the world) 

  • Decontamination (go through the area to decontaminate you) 

  • Access zones 

  • Anterooms 

  • Airlocks (the room where air one side is separated, doesn't mix w air form others) 

• Facility Practice & Procedures 

  • Handling, storage, management (things to do not contaminate) 

what does each BSL do?

• Each BSL adds onto each other (BSL 1:1, BSL 2: 1+2, BSL 3: 1+2+3) 

What is BSL 1

• variable biological agents not known to cause disease in healthy adult humans 

What is BSL2

• bio agents & toxins cause disease in humans of varying severity 

What is BSL 3

•  indigenous or exotic bio agents with a potential for respiratory transmission and is potentially lethal 

What is BSL 4

•  high life threatening disease that may be transmitted via the aerosol route, which there is no available vaccine or therapy 

Covid is what biosafety level

BSL3

Aseptic technique

minimize risk of contamination to cells + urself

• Sterile work env 

• Good personal hygiene 

• Sterile reagents + media 

• Sterile handling 

What is the BSC layout?

Ventilation in one direction and goes up instead of coming at u 

BSC types

Class I – similar to fume hoods, protect individual 

Class II – BSL-1 to BSL-3, protect individual + culture 

Class III – BSL-4, highest available protection

What are good practices to keep your BSC clean?

• Work area uncluttered 

• Regular disinfection 

• Regular equipment cleanup & sterilization 

• Maintaining equipment sterility 

• Maintaining reagent sterility

When your contaminant is bacteria what are the visual signs vs chemical signs

visual: cloudy sulture (turbidity) [if floating]

microscpic: granules beyween cells

chemical: decr pH

When your contaminant is mold what are the visual signs vs chemical signs

visual: turbidity

microscopic: spherical, bud off

chemical: incr pH

When your contaminant is yeast what are the visual signs vs chemical signs

microscopic: waspy fillaments, spore clumps

chemical: incr pH

what are the signs of viruses?

mainly affect cultures from species of their host, hard to detect, can be dangerous

what are signs of mycoplasma (organisms not bacteria)

hard to detect until severe

chronic and alters cell activity