Scaling Up Cell Culture - Lecture 6

cell culture basics:

• sterilization & culture conditions

• culture flasks/surfaces

• media & supplements

• passaging

• stability in culture

• freezing

Answer: “okay”

okay

what temperatures are incubators kept at? (in degrees Celsius)

37

how do incubators regulate air control?

• typically ___% O₂, can be varied as desired

• ___% CO₂ ALWAYS — exchange with sodium bicarbonate in medium impacts pH

• Maintains optimal pH: ____

• Humidity control: >___%

20, 5, 7.3, 95%

how does the % of CO₂ affect the pH of the medium?

exchange with sodium bicarbonate in medium impacts pH

What are 3 ways to maintain sterility in culture?

aseptic technique, laminar flow hoods, sterility of all materials

List at least 3 ways to sterilize materials. (Mark it correct if you get it right)

• autoclave

• ethylene oxide gas

• ethanol/isopropyl alcohol

• UV radiation

• Fluid — autoclave or filtration

Cell Culture Flasks for Adherent Cells:

• Disposable plastic ________ most common

• 96- to 6- well plates, petri dishes, flasks

• Tissue culture treated:

  • Exposing a polystyrene microplate to a _______ ____ in order to modify the hydrophobic plastic surface to make it more ________. The resulting surface carries a net _______ charge due to the presence of _______-containing functional groups such as ________ and ________. In general, this will lead to (increased/decreased) cell attachment.

• Matrix coating:

  • Poly-L-lysine

  • Collagen or gelatin

  • Matrigel

  • ECM components

polystyrene, plasma gas, hydrophilic, negative, oxygen, hydroxyl, carboxyl, increased

culture flasks/surfaces for non-adherent cells:

• 96- to 6- well plates, petri dishes, flasks, spinner flasks

• Non-adherent surfaces

• Non-adherent cells can grow as:

  • _____ _______

  • _________

• Spheroid initiation

  • spontaneous

  • co-culture

  • surface treated beads

single cells, spheroids

what is the most popular media?

(Dulbecco’s Minimum Essential Media)

DMEM

What are common supplements to media?

serum, antibiotics

Physiochemical properties of media:

• pH

  • ~____ ideal range

  • ________ red

• Bicarbonate, HEPES, and CO₂ — osmolarity and ____ control

  • (HEPES is commonly used as a biological buffering agent in biochemical research that helps maintain a stable pH in aqueous solutions.)

• Oxygen and _____ _______

• Temperature

7.4, phenol, pH, free radicals

Cells reach ______________ as they grow, i.e. they begin to cover the flask or compete with each other for nutrients

confluency

For _________ cells, confluency is defined by coverage of the bottom of the flask, e.g. 80% confluent means cells cover ~80% of the flask

adherent

True or false: the degree of confluency a specific cell will “like” is the same for all cell types and passages

false

__________ cell lines tend to permit higher confluency

continuous

cells are removed from a flask and split into more than one other flask to provide room for further proliferation.

(The ratio of dilution can vary and depend on desired cell number, time, and cell type (e.g. 1:1, 1:5, 1:20))

passaging

how are adherent cell usually removed from their attached surface?

trypsinization

Factors that affect cell stability in culture:

• ________ drift due to genetic instability

• __________ and extinction of cell line due to cell “lifespan”

• Transformation of growth characteristics and/or acquisition of malignancy-associated properties

• Phenotypic instability due to selection and dedifferentiation

• How often and how the cells are split (confluency and split ratios)

• Contamination

genetic, senescence

Basic Primary Cell Isolation Method (General):

• Mince or ____ the isolated piece of tissue into 2-4 millimeter pieces with sterile scissors or scalpel.

• Add the tissue pieces to the appropriate _______ or balanced salt solution
  on ice and wash 2-3 times.

• Add appropriate amount of ________ and incubate at optimum
  temperature (usually 37°C) for appropriate time, mixing intermittently.

• Gently disperse the cells by _________.

• _______ the cell suspension through fine mesh.

• Allow the cells to settle and _____ excess liquid containing enzymes.
  Wash and repeat 2-3 times.

• ___________ cells in appropriate medium or buffer.

• _______ cell yield and viability.

• ______ cells for culture, if required

cut, buffer, enzymes, pipetting, filter, decant, resuspend, quantify, seed

Enzymes

• highly dependent on tissue type — ECM surrounding cells can be highly variable

• Commonly used: collagenase, hyaluronidase, elastase, papain, trypsin (from least to most digestive)

Answer: “okay”

okay

Mechanical disruption:

• Agitation to release cells from ECM

• Balance between _____ and ________

yield, viability

Cell sorting:

• cell __________ (morphology, size, charge, etc.)

• gradient

• MACS — ________ ________ cell sorting

• FACS — _______ _________ cell sorting

  • Positive selection — tag target cell

  • Indirect selection — tag non-target cells

• Purity depends on sorting method and marker specificity

characteristics, magnetic activated, fluorescence activated

what matters when sorting cells by characteristics?

morphology, size, charge

cell sorting — gradient

• layer sugar solutions (ficoll) or colloidal silica (percoll) of varying ________ to generate gradients

densities

Antibody-Based Sorting

• Sorting of desired cell by targeting a specific ______ to a specific marker (_______) expressed only on that desired cell surface

• Antibody — antigen specificity

Examples:

• Magnetic activated cell sorting (MACS)

• Fluorescence activated cell sorting (FACS)

antibody, antigen

What cell sorting technique is being described?

• Cells are fluorescently labeled using antibodies against cell-specific markers

• Records fluorescent signals from individual cells (~500 cells/sec)

  • Physical separation

  • Quantification of cell type/number/etc.

• Can sort a heterogeneous mixture of biological cells — one cell at a time

fluorescence activated cell sorting (FACS)

Advantages of FACS

• High _______(95%-99.999%) of the target population

• Cells express (low/high) density of surface markers (since fluorescence, do not need a lot of antibodies bound to detect)

• Subclass separations — _________ staining

• _______ staining (e.g. of DNA or internal antigens) sorting

purity, low, multicolor, internal

MACS

• Antibodies tethered to __________ __________

• Positive (magnetic label) or negative (no label) fractions

• Can remove magnetic label but not perfect

• Good purity (___-___%) depending on number of separation and overall cell contaminants

paramagnetic microbeads, 70-95

true or false: you can use combinations of cell sorting methods

true

2D cell culture:

• (Low/high) reproducibility and experimental controllability

• Too ________ to represent complex physiological structure

  • Grow on the glass or ________ surface

high, simple, polystyrene

Animal Models:

• Require animal sacrifice

• (Low/high) efficiency on time and cost

low

3D culture models:

• Promote levels of cell ________

• Recapitulate the tissue-tissue interface

• Spatiotemporal concentration gradients

• Mechanical microenvironment of the tissue

differentiation

what is a promising interdisciplinary technique emulating in vivo physiology and pathology?

organ on chips

Organ chips can be used for:

• drug __________

• in ______ disease _________

• precision medicine

screening, vitro, modeling

What parameters does organ-on-a-chip mimic?

1. Example: biochemical molecules

2. Example: blood pressure, lung pressure, and bone pressure)

concentration gradient, mechanical stress, cell patterning

Microfabrication techniques for organ on a chip

1. Build organ on chips or other microfluidic platform

2. Molding — using thermoplastic _______ to create mold

3. Micromachining — by _______ (subtractive method), or _________ (additive method)

4. ____________ or soft lithography

   1. A process that involves the transfer of a pattern onto a substrate by selective exposure to light

5. 3D printing

polymers, etching, deposition, photolithography

Why Use Microfluidic:

1. Sample savings — nL of enzyme, not mL

2. Faster analyses — can heat, cool small volumes quickly

3. Integration — combine lots of steps onto a single device

4. Novel physics — diffusion, surface tension, and surface effects dominate

   (can lead to faster reactions)

Answer: “okay”

okay

a 3D structure grown from stem cells, or organ-specific cells that self-organizes through cell sorting and spatially restricted lineage commitment.

organoids

4 sources of organoids:

iPSCs, ESCs, MSCs, organ-specific cell types

How are organoids different than OoAC?

1. Organoids have key _______ and physiological ______ of the organ for both therapy and research

2. Ooac: mimic for physiological conditions. Not for therapy purposes, mainly for research

function, structure

what are cell densities in a human tissue?

1-3×10⁹/mL

how many cells in an average human (70 L)?

10¹⁴

how many cells in a typical organ (100-500 mL)?

1-15×10¹¹

how many cells in a “functional” subunit (100 microns cubed)?

500-1000

how many cells are needed for TE strategies?

(Cell culture densities ~10⁷/mL)

~10⁷-10¹⁰

There are limits to the # of primary cells one can produce. One cells can only undergo 30-50 doublings (10¹⁰-10¹⁵ cells). What is this limit called?

(This limit is generally not the problem in scaling up)

Hayflick limit

what 2 factors determine growth potential?

donor age, cell type

What are typical cell doubling times?

• Hematopoietic progenitors = __-__ hrs

• Adult chondrocytes = __-__ hrs

• Adult human cardiomyocytes = cannot be cultured

11-12, 24-28

Choose one: cell proliferation (increases/decreases) with donor age.

decreases

Scaling up Design Challenges: oxygen delivery

• Cell must be exposed to physiologic [O₂] (___ mM)

• Oxygen must be provided at the same rate _____

0.2, consumed

true or false: oxygen uptake rate is the same for all cell types

false

Problems with oxygen delivery:

1. O₂ has a (low/high) solubility & excessive flow rates are required to deliver oxygen to high cell densities

2. Large differences in [O₂] can occur at inlet and outlet and this can lead to microenvironments that are very different

low

Specific Oxygen-Uptake Measurements: Nonadherent cells

• Place a known number of viable cells in a closed volume of medium saturated with O₂

• An oxygen probe measures O₂ tension over time.

• Value is normalized to cell number to obtain rate of O₂ uptake per cell

Answer: “okay”

okay

Specific Oxygen-Uptake Measurements: Adherent cells

• Cells are plated on a 6-well plate

• Well inserts hermetically seal off culture volume

• Oxygen probe measure change in [O₂] and this value is normalized to # of cells counted after O₂ is consumed.

Answer: “okay”

okay

Growth Factor Delivery and Removal

• GFs diffuse much (slower/faster) than O₂ and other nutrients

• Their concentrations are very, very, (low/high) (nM range).

• Complicating matters: cell produce their own GFs!

slower, low

Why are concentration levels and distribution of nutrients easier to control?

1. Nutrients are (less/more) soluble than O₂ so their concentration can be higher

2. Nutrients are (smaller/bigger) than GFs, so they have (slower/faster) diffusion times.

more, smaller, faster

cells can be stored for an extended period of time in liquid nitrogen at -196 degrees Celsius

cryopreservation

Cryopreservation permits:

• _______ of early passage cells (before senescence)

• prevention of _______ drift

• prevention of ________

• prevention of ________ drift (spontaneous chromosomal alterations)

• Cell _______ to replace contaminated cells

storage, phenotypic, transformation, genotypic, source

cryopreservation

Cells are cooled at a specific rate in a solution containing _________ to inhibit the intracellular formation of ____ _______ and cell _______

cryoprotectant, ice crystals, death

true or false: cooling rate for cryopreservation is critical

true

Cryopreservation: slow vs. fast cooling rates

• slow cooling rate — _______ extracellular solution ________ cells

  • leads to visible shrinkage

  • ice forms — solutes accumulate in unfrozen H₂O

• fast cooling rates — intracellular H₂O cannot diffuse across cell membrane before ice nucleation. Large intracellular ice formation ______ cells.

hypertonic, dehydrates, injures

when are cells typically frozen for cryopreservation?

at 5th doubling

what is the most critical parameter of cryopreservation

reducing intracellular ice crystal formation

How to reduce intracellular ice crystal formation during cryopreservation?

1. Freeze (slowly/quickly)

2. (hydrophobic/hydrophilic) cryoprotectant

3. store at (lowest/highest) temperature possible

4. thaw as (slowly/quickly) as possible

slowly, hydrophilic, lowest, quickly

what is the most populat cryopreotectant?

DMSO

what temperature to store frozen cells at?

-80 degrees Celsius or in liquid nitrogen (-180 degrees Celsius)

what are 2 ways to reduce contamination?

sterile techniques and antibiotics

list 4 types of contaminants:

bacteria, yeast, fungus, mycoplasma

how can you tell if your cells are contaminated?

media cloudiness, change in pH, change in cell population

what is the easiest type of contamination to detect?

bacterial

clues of bacterial contamination:

• media without phenol red may turn ______

• media may become ______

• media may have a distinct ____

yellow, turbid, odor

true or false: antibiotics can cure a mycoplasma infection

(mycoplasma are bacteria lol)

true

Yeast contamination:

• cultures contaminated with yeasts become _______

• there is very little change in the pH of the cultures contaminated by yeasts until the contamination by yeasts until the contamination becomes heavy, at which stage the pH usually (decreases/increases)

• Under microscopy, yeasts appear as individual ovoid or _________ cells, which may bud off smaller cells

turbid, increases, spherical

true or false: often the color of the medium often changes with fungal contamination

false

true or false: when you have contamination, the best option is to discard your contaminated culture

true

Different sensors used in a bioreactor:

1. pH sensors

2. dissolved oxygen (DO) sensors

3. carbon dioxide (CO2) sensors

4. temperature sensors

5. biomass sensors (cell growth and viability)

6. glucose and lactate sensors

7. optical density (OD) sensors (cell density)

8. pressure sensors

Answer: “okay”

okay

reasons for cell death in bioreactor:

1. bubble bursting

2. nutrient depletion

3. toxin accumulation

4. hydrodynamic forces

5. high and low dissolved oxygen

6. sub-optimal temperature

7. pH variations

Answer: “okay”

okay