tissue 3

what is the goal of cell culture (2)

1. maintain or expand a population of cells, & the single most important consideration in cell viability

2. freeze, store, & recover cells provides an essential safeguard against losing a cell line to contamination, incubator malfunction, an error on the part of the investigator, or avoid senescence & genetic drift

cell/tissue culture

process or technique of making body tissue grow in a culture medium outside the organism

cell culture

complex process by which cells are grown under controlled conditions, generally outside of their natural environment

organoid culture

tissue culture method to grow functional 3D organoids from a group of cells & combination of various biochemical factors

organ culture

development from tissue culture methods of research, the organ culture can accurately model functions of an organ in various states & conditions using the actual in vitro organ itself

why is the study of cells is carried out using cell culture (3)

1. cells can be obtained in large quantities

2. most culture contain a single type of cell

3. many different types of cells can be grown in culture

why is cell culture significant (3)

1. study of diverse cellular processes

2. primary testing ground for the study of development &disease

3. advancement of knockout technology & drug discovery

cell theory (4)

matthias schleiden, theodor schwann, rudolf virchow

1. all organisms are composed of one or more cells

2. the cell is the structural unit of life

3. cells arise only by division from a pre-existing cell\

4. cells contain genetic info passes to next generation

primary culture

started from cells, tissues, or organs taken directly from an animal

organ culture

maintenance of tissues, whole or parts of organs in a way that may allow differentiation & preservation of the architecture &/or function

explant

an excised fragment of an organ which usually retains some degree of tissue architecture

tissue culture

maintenance of a tissue or fragment thereof in a way that may allow differentiation & preservation of the architecture &/or function

cell culture

maintenance of dispersed cells

monolayer

single layer of cells growing on a surface

subculture

transplantation of cells from one culture to another (passaging)

cell line

arises from the primary culture at the time of the first subculture - finite life span

primary cell lines (3)

isolated directly from animal or plant tissue

not immortalized

finite doublings, with potential to become immortalized

continuous cell lines

when a finite cell line undergoes transformation & acquires the ability to divide indefinitely

transformed cell lines

from tumors or are transformed either spontaneously or deliberately

suspension growth

suspension cells

suspension cells (3)

1. able to survive & proliferate without attachment to the culture vessel

2. cells from blood, spleen, bone marrow

3. various shapes but generally rounded

suspension cells advantages (2)

1. large numbers

2. ease of harvesting

adherent growth

adherent cells

anchorage-dependent

anchorage-independent

adherent cells (3)

1. grow in monolayer, attached to surfaces of culture vessels

2. from ectodermal or endodermal embryonic cells (fibroblasts, epithelial)

3. various shapes but generally flat

adherent cells advantages (2)

1. spread on surfaces such as coverslips

2. easy for microscopy or other functional assays

anchorage dependent cells

require attachment to the surface for cell proliferation

anchorage-independent cells

property of transformed cells

maintenance

examine daily cultures, observe morphology, the color of the medium and the density of the cells

feeding

suspension cultures are fed by dilution into fresh medium

adherent cultures either replace old with fresh medium

splitting

done at 75-100% cell confluence, prevents the increase in the cell death and prevents the decrease mitotic index (trypsin)

preservation & storage (2)

1. cells can be damaged by ice crystals, alterations in the concentration of electrolytes, dehydration, & changes in pH

2. cryptoprotective agent which lowers the freezing point (glycerol or DMSO) is +

how do you isolate cells for culture (4)

1. remove tissue

2. mince or chop

3. digest with proteolytic enzymes

4. place in culture

important features/requirements of tissue/cell culture (5)

1. cells

2. sterile environment to work with cells - lab hood

3. controlled environment for cell growth - incubator

4. something for the cells to grow on - flasks, dish

5. food - medium

common cell lines (4)

1. 3T3 - fibroblast (mouse)

2. HeLa - cancer epithelial cell (human)

3. CHO - ovary (chinese hamster)

4. HEK293 - human embryonic kidney-derived epithelial cells

can u buy cells

cell & tissue banks

why are cell lines/strains cloned & immortalized (4)

1. well characterized & stable

2. allows valuable comparisons from lab to lab

3. can study features of particular cells that are especially difficult to isolate & culture

4. great for tissue engineering research, not for product dev

culture environment - substrate (4)

1. solid

2. semisolid

3. liquid

4. feeder layers

solid culture substrate (4)

1. glass

2. plastic (treated polystyrene)

3. coat plastic with ECM components

4. cells usually grown in monolayers, then passaged & expanded

semisolid culture substrate (3)

1. agar/ECM based matrix

2. collagen gel cellulose-sponge

3. aids tube formation

liquid culture substrate

simply suspension cultures

feeder layers culture substrate

layers of cells/tissues which are treated (irradiation/chem) to stop them from proliferation to form the basis on which other cells such as ES cells can be cultured

various colony morphologies (5)

1. entire

2. undulating

3. erose

4. lobate

5. filamentous

culture environment - gas

O2 & + CO2 (5% dependent on NaCO3 level in medium)

O2 culture gas (2)

1. atmospheric tension but some organ culture require increased levels

2. others grow better with reduce oxygen levels (chondrocytes)

CO2 culture gas (2)

1. for bicarbonate buffered media, tension regulates pH

2. radioactively labeled CO2 is incorporated into ribose

culture environment - media (6)

1. choice of media is cell-type specific & empirical

2. should provide nutrients, buffering capacity, isotonic, sterile

3. balanced salt solutions, simple mix of salt + glucose

4. dependent on conditions vary in bicarbonate concentration

5. Hanks BSS - used with air, Earles BSS - used with 5%CO2

6. tissue disaggregation - divalent cations omitted

complete media (4)

1. wider range of ingredients needed to support survival & prolif (7.4 pH)

2. bicarbonate buffer

3. HEPES

4. osmolality

media (4)

most contain:

1. AA

2. salts

3. glucose

4. serum

serum (3)

1. liquid left over after fibrin & cells are removed from blood

2. calf, fetal bovine, horse used

3. tole is not clear

major functions of serum (3)

1. basic nutrients - in solution & protein bound

2. supply of growth factors & hormones

3. contains factors promoting attachment & spreading on artificial surfaces protease inhibitors

growth factors & cytokines - serum (6)

1. PDGF

2. EGF

3. IGF

4. FGF

5. IL-1

6. IL-2

proteins - serum (4)

1. albumin

2. fibronectin

3. transferrin

4. a2-macroglobulin

hormones - serum (4)

1. insulin

2. hydrocortisone

3. thyroxine

4. triiodothyronine

vitamins - serum (2)

1. vitamin A

2. folate

lipids - serum (2)

1. cholesterol

2. phospholipids

misc. - serum (4)

1. AA

2. salts/inorganics

3. carbs

4. urea

5. polyamines

6. fetal bovine

disadvantages of serum (4)

1. comp is ill defined

2. high variability

3. viral & prion contamination

4. protein content complicates purification of cell products from medium

serum alternatives

replacing essential components for particular cell type: highly selective, effort, limited to few cell types

disadvantages of serum-free (4)

1. multiplicity of media (each cell needs diff media)

2. selectivity (may select a sub-lineage in cell line)

3. reagent purity (removes protective, detoxifying action of some serum proteins)

4. cell proliferation (slower growth, fewer gen)

what is cell culture

growing cells outside the organism

why is so much research carried out on cultured cells (5)

1. cultured cells can be obtained in large quantity

2. most only contain a single type of cell

3. many different cell activities can be studied

4. can differentiate in culture

5. respond to treatment

what has been a primary goal of cell culturists

develop defined serum-free media that supports the growth of cells

what does optimal temp depend on (4)

1. body temp of animal

2. regional variation

3. usually 36.5 C

4. can lead to entrainment of cell cycle

what is contamination (5)

1. bacteria

2. yeasts

3. fungi

4. molds

5. mycoplasma

what are the sources of contamination (3)

1. sterile technique

2. contaminated cells

3. contaminated air supply

what defines contamination (4)

1. sudden pH change of media (decrease = bacteria Y, increase = fungal P)

2. cloudiness of media

3. change in culture appearance

4. fluorescence

how to prevent contamination (2)

1. antibiotics & fungicides in media

2. good sterile technique

tissue culture flasks (3)

1. sterile - polystyrene

2. treated to promote nonspecific cell attachment

3. coat flask to enhance cell growth

biological safety cabinet

laminar flow hood

ensures cell are in contact with decontaminated air

laminar flow

unidirectional air moving at a steady velocity in parallel lines

primary barriers

protect the material being manipulated within the hood from worker generated or environmental sources of contamination

vertical hood

best for working with hazardous organisms since the aerosols that are generated in the hood are filtered out before they are released into the surrounding environment

horizontal hoods

designed such that the air flows directly at the operator but best protection for cultures

what are some of the important physiochemical properties of medium (7)

1. pH

2. CO2

3. buffering

4. oxygen

5. osmolality

6. temperature

7. viscosity

buffering of medium

HEPES - maintain pH

CO2

5-10%

in gas phase appears as dissolved with HCO3-

indicators

phenol red

what are some medium supplementation anticontaminants (2)

1. antibiotics

2. antifungal

aminoglycoside antibiotics G418 Genectin

micromonospora rhodorangea

binding ribosome stopping protein synthesis

blocks peptide synthesis by stopping elongation step

resistance - neo gene

used to select genetically engineered cells

trypsin (4)

1. pancreatic serine protease

2. carboxyl group of arginine & lysine

3. releasing cells into suspension from monolayers

4. aided by EDTA which loosens Ca2+ dependent cell-cell adhesions

what are the exceptions to adherent monolayers (3)

1. hematopoietic cells

2. transformed cell lines

3. cells from malignant tumors

passing the cells

detaching & reattaching

splitting the cells

diluting the concentration

what does the formulation of a cell line from primary culture imply (4)

1. increase in total # of cells over several gen

2. predominance of cells or lineages with capacities for high growth

3. degree of uniformity in cell pop

4. ex: subculture of HFFs by Apigraf

what is aseptic technique

to prevent contamination from pathogens

applying strictest rues to minimize risk of infection

why 70% alcohol

can cross over cell membrane attacking entire cell line & killing bacteria

what are the benefits of using 70% alc (3)

1. coagulation of surface proteins are slower allowing alc to enter cell

2. contains water which is essential in denaturing process of proteins

3. increases potency of antimicrobial properties

primary culture

heterogenous pop of cells

purity of primary culture

one cell type will outgrow the others - fibroblasts

selective inhibitors

1. natural

2. genetically aided

natural selective inhibitor

replace L-valine in medium with D-valine

cells with D-amino acid oxidase grow preferentially

genetically aided selective inhibitors

transfection

transfection

supply subpopulation of cells with resistance to a drug, then add that drug to culture medium

cell density - centrifugation

can speed up process using centrifugal elutriation

antibody based techniques - cell separation

identify an antigen

immune panning

magnetic sorting

cell characterization

It is crucial to routinely characterize your cells to make sure you know what you are working with

differentiation

process leasing to expression of phenotypic properties characteristic of the functionally mature cell in vivo

2D

when cells are grown on the flat surface of a dish

3D

grown in a 3D matrix consisting of extracellular materials

better suited to study cell-cell interactions

“requirements” for cell line characterization (6)

1. confirmation of species

2. correlation with tissue of origin

3. cell transformation?

4. cross contamination?

5. prone to genetic instability?

6. features unique to that cell line/strain