Labs

Question 1

What is PBS and how do we make it

Answer 1

phosphate buffered saline

one of the most used buffers in bioengineering
->similar tonicity to tissues, and cytoplasmic (cellular solution conditions)
-> avoid osmotic shock and any potential cell/tissue damage.

prepare a PBS solution using the
- weak acid salt (monosodium phosphate) and its conjugate base (disodium phosphate)
- adjusted to a final pH value to 7.4 using hydrochloric acid (HCl) or sodium hydroxide (NaOH).

NaCl, Na2PO4, KH2PO4, KCl

Question 2

How does a pH meter work

Answer 2

measuring the hydrogen ion concentration in a solution (pH, defined as the negative logarithm of the hydrogen ion concentration)

This glass electrode is sensitive to hydrogen ions and generates a voltage that varies with the pH of the solution it’s in contact with. This voltage is compared to a reference voltage from a reference electrode, usually filled with a stable electrolyte solution like 3M KCl.

Question 3

Decellularized plant scaffolds characteristics, advantages and challenges

Answer 3

Characteristics of Decellularized Plant Scaffolds:

• Physical resemblance to human organs.
• High superficial surface area.
• Excellent water transport and retention capabilities.
• Pre-existing vascular networks.
• Interconnected porosity.
• Range of mechanical properties.

Advantages of Plant-Derived Scaffolds:

• Unique and superior biocompatibility.
• Low production costs.
• Free from ethical concerns and supply limitations.
• Suitable for large-scale production and research.

Challenges with Decellularized Plant Scaffolds:

• Immature decellularization standards and methods.
• Limited research on the biocompatibility of plant extracellular matrix (ECM).( early stages of research)

Question 4

scCO2 decell

Answer 4

supercritical carbon dioxide (scCO2) decellularization involves using scCO2, a fluid state of carbon dioxide where it behaves as both a gas and a liquid, to remove cellular material from plant leaves

Hexane (removes non-polar to go faster and more thorough) with PBS to stabilize and rinse, place in cylinder to dissolve with scCO2 and paracetic acid to sterilize.

Question 5

bleach decell

Answer 5

bleach (remove proteins) and an alkaline agent like NaOH (saponification) heated and mixed with tissue

After, plant material is washed with PBS to remove any residual chemicals before lyophilization (freeze drying for storage) and storage.

Question 6

Picogreen assay

Answer 6

The PicoGreen assay is a fluorescence-based quantification method for double-stranded DNA (dsDNA). It uses a PicoGreen dsDNA quantitation reagent that selectively binds to dsDNA. When bound, the PicoGreen dye becomes highly fluorescent and the intensity of this fluorescence is proportional to the amount of dsDNA present in the sample. Set the microplate reader to specific wavelength to excite it and to absorbs from its subsequent emission.

Use standard DNA tube with PicoGreen dye to measure and create a standard surve.

Question 7

BioDrop assay

Answer 7

a BioDrop spectrophotometer quantifies DNA by measuring the UV absorbance at 260 nm, which is then used to calculate the concentration of DNA in the sample based on the known absorbance properties of nucleic acids.

we used 2uL

Question 8

What is UView dye for

Answer 8

UView dye is a nucleic acid stain used in agarose gel electrophoresis. It is designed to intercalate with DNA and RNA, allowing you to visualize the nucleic acid bands under ultraviolet (UV) light after electrophoresis.

Question 9

How does gel electrophoresis work basics

Answer 9

DNA is negatively charged due to the phosphate groups in its backbone, which carry a negative charge at neutral and alkaline pH levels commonly used in gel electrophoresis. This negative charge causes DNA to migrate towards the positive electrode (anode) during electrophoresis.

all have a similar charge-to-mass ratio because the negative charge is uniformly distributed along the phosphate backbone of the DNA.
primary factor determining the rate of migration through the gel is the size (length) of the DNA molecules, rather than differences in charge.

(Larger DNA fragments have more nucleotides, so they carry more overall negative charge. This means they are subject to a greater total electrical force in an electric field.Greater Mass Slows Movement: However, these larger DNA molecules also have more mass, which makes them move more slowly through the gel matrix. )

Smaller DNA fragments move through the gel matrix more easily and therefore travel further than larger ones in a given amount of time.

Question 10

What are hydrogels and how are they typically composed?

Answer 10

Hydrogels are polymers that are cross-linked and highly swollen in water. They are often comprised of naturally occurring polymers.
Insoluble networks containing large amounts of water

Question 11

What are examples of a hydrogel in the human body, and what are their properties?

Answer 11

Skin is an example of a hydrogel in the body. It is strong, resilient, and has extraordinary healing capabilities, despite being primarily made of water.

Cartilage is a hydrogel with astounding mechanical properties, like strength and elasticity, capable of handling large extensions and high loads in bone joints.

Question 12

What are some potential uses of synthetic hydrogels?What is a common issue with many synthetic hydrogels?

Answer 12

Synthetic hydrogels can be used in drug delivery, as extracellular matrix mimics, and in tissue engineering.

Many synthetic hydrogels have poor mechanical properties, undergoing irreversible damage upon high loadings.

Question 13

What are two essential attributes for hydrogels used in tissue engineering and how can hydrogels achieve these?

Current situation?

Answer 13

Hydrogels for tissue engineering need to adhere to cells and exhibit compatibility with their surroundings, as well as have mechanical strength.

Achieved by combining two different polymeric networks: one for biocompatibility and the other for mechanical strength.

bulk polymers (synthetic elastomers) are much stronger than hydrogels and hydrogels are weaker than cartilage

Question 14

What does the sol-gel transition temperature indicate in gelatin?

Answer 14

The transition temperature marks when gelatin molecules reform from random coils into triple-helices acting as physical cross-links below 35°C; above this temperature, gelatin behaves as solutes in solution.

Question 15

What is vial inversion gel for

Answer 15

Visualize sol-gel transition in gelatin

Question 16

What happens to gelatin’s complex viscosity during the sol-gel transition?

Answer 16

The complex viscosity of gelatin increases as it transitions from liquid (sol) to gel over time.

Question 17

What is alginate and how does it work

Answer 17

Alginate (or alginic acid) is a naturally occurring polymer found in the cell walls of brown algae. It is water soluble, wherein the cells it binds with water to form a viscous sticky gum. It is technically a copolymer, with blocks (segments of repeating units) of both ß-D- mannuronate (M) and its C-5 aptamer α-L-guluronate (G).

Question 18

How does alginate cross-linking work

Answer 18

Crosslinking involves using divalent calcium ions (Ca^2+) to form non-covalent bonds between alginate polymer chains. The calcium ions can bind reversibly with the acid groups in the alginate’s G units, creating interchain bonds. This process replaces the monovalent sodium ions (Na^+) in the alginate, which can’t act as crosslinkers, with calcium ions that can form bonds between multiple chains, thus creating a crosslinked network.

Question 19

Cross-linking polymers

Answer 19

Crosslinking polymers refers to the process of chemically or physically connecting the polymer chains together, creating a network structure. This crosslinking enhances the strength, stability, and various properties of the polymer, such as its elasticity and resistance to heat and chemicals.

Question 20

What types of materials do we have in a stress strain sense

Answer 20

brittle material, breaking at low strain, but requiring a relatively high force (or stress). (steep slope)

tough material, exhibiting strength but also relatively high elongation. (steep with plateau)

soft material, being highly extensible, but breaking with relatively little force. (not v sleep, low plateau)

Question 21

What is stress and strain, curve

Answer 21

stress = sigma = F/A0
Strain = lambda = L stretched / L original

Question 22

How does mycoplasma detection work

Answer 22

researchers design PCR primers that are complementary to mycoplasma DNA, so when mycoplasma is present in the sample, the PCR amplification will produce a product of the expected size, which is typically around 500 bp. This is a common strategy for detecting mycoplasma contamination because it provides a specific and reliable way to identify its presence in cell cultures.

Question 23

How does sds quantification work

Answer 23

In our SDS quantification protocol, a standard curve is constructed using diluted SDS solutions, and then samples containing SDS are mixed with Methylene Blue and Chloroform (solubilize sds and methylene), incubated, and their absorbance at 650 nm is measured using a spectrophotometer. The concentration of SDS in the samples is determined by comparing their absorbance to the standard curve, allowing for quantification of SDS content.

the dye and sds form a complex which changes the absorbance

Question 24

scCO2 mammalian

Answer 24

osmotic shock, trypsin for protein wash, PBS,

scCO2 for decell

wash again

scCO2 for sterilization

Question 25

SDS decell mammalian

Answer 25

use PBS wash, SDS, antibiotics for bac, detergent non-ionic (triton 100x),

DNase and RNase, sterilize with paracetic acid

Question 26

Osmotic-based

Answer 26

Freeze thaw, osmotic shock, antibiotics, enzyme to remove proteins

Question 27

Bradford assay

Answer 27

The Bradford assay is a protein quantification method that relies on Coomassie Blue dye binding to proteins, resulting in a color change proportional to protein concentration, turning them from brown to blue, which is measured spectrophotometrically and compared to a standard curve for accurate protein quantification.

Question 28

SDS-Page electrophoresis

Answer 28

Coats prots in negative charge and denatures them using SDS, removes disulfide bonds using reducing agent DTT or B-mercaptoethanol

They are linearized and go through gel based on size alone, not the individual charge of the protein itself or its shape (so + proteins and - proteins all go in same direction)

Question 29

What buffers and gels for dif electrophoresis

Answer 29

TGX and polyacrylamide for sds page, laemmeli for dye

TAE and agarose (1% protein, 2% mycoplasma 500 bp) UView dye for gel electro

Question 30

Gel-MA

Answer 30

DLP SLA

Methacrylated-gelatin (Gel-MA) is a modified form of gelatin in which methacrylate groups (functional groups containing a double bond) are attached to the gelatin molecules. These methacrylate groups make the gelatin responsive to light and enable it to form a gel structure upon exposure to specific wavelengths of light.

Photoinitiator (e.g., Irgacure or eosin Y):

A photoinitiator is a compound that initiates a chemical reaction when exposed to light, typically ultraviolet (UV) or visible light.

A photosensitizer is a compound that enhances the sensitivity of a system to light. It absorbs light energy and transfers it to other molecules, often promoting chemical reactions.

Question 31

How to make alginate beads

Answer 31

prepare alginate and water, then add dye
drop beads in CaCl2 solution while stirring.

The crosslinking of alginate by calcium or barium ions causes the alginate solution to solidify rapidly into spherical beads. These beads are essentially gelatinous capsules where the alginate molecules are linked together by the ionic bonds with the divalent metal ions.

Question 32

what does adding surfactant do to alginate beads

Answer 32

Adding surfactant makes the beads smaller, bc there is less surface tension allowing the beads to be large

Question 33

Crosslinking ion variation

Answer 33

Calcium ions are versatile and widely used for their biocompatibility and efficiency, while barium ions may be preferred when enhanced rigidity and mechanical strength are needed.

Question 34

what is dye loading capacity and efficiency

Answer 34

Dye loading capacity refers to the maximum amount of dye that a material or substrate can absorb or incorporate.
Dye loading efficiency measures how effectively a material or substrate can uptake and retain a dye compared to the available dye in a solution, typically expressed as a percentage.

Question 35

define deo terms
factor, response, type of variable, levels, trial, treatment

Answer 35

Factor: variable studied in DOE
Response: measures outcome of interest
Controlled variables: constant
Lurking: uncontrolled
Levels: values which variables can have
Trial : experimental run
Treatment: combination of all factors (ex.: treatment = temp and time)

Question 36

types of DoE (4)

Answer 36

Types:
- Factorial: all combinations of factors run (2^k) -> 2 levels, k = factors
o a design in which you run all possible combinations of factor levels
- screening: design that is used to identify the most important factors and interactions
- response surface: design that is used for optimizing a product, process, or system
- custom: design based on the model that you need to estimate and your experimental goal and budget

Question 37

center point doe

Answer 37

Center point: refers to a specific combination of factors or variables at their midpoints
- Test for lack of fit