Cell Culture- Master Deck

Question 1

Major function of bioreactors:

Answer 1

Devices used extensively in tissue engineering to ENABLE the fabrication of 3D artificial tissue and SUPPORT the growth, maturation, and development of artificial tissue engineering during controlled in vitro culture

Question 2

Effects of perfusion on cell seeding

Answer 2

Cells will penetrate the whole scaffold and perfuse more homogenous

Question 3

Methods for estimating number of cells in a reaction

Answer 3

1. Glucose or oxygen consumption
2. Specific product: (hepatocytes monitor urea, fibroblasts produce collagen, general: lactate…yeast produces ethanol)

Question 4

Role of physical conditioning

Answer 4

1. SYNTHESIS & HIERARCHY: Physical forces have been shown to be potent regulators of cell physiology in vivo, and modulate ECM synthesis and organizations at various hierarchical levels- from molecules to whole tissue/organs
2. DIFFERENTIATION: Mechanically generated signals play a significant role in differentiation of progenitor cells into different lineages
3. ORGANIZATION: Effective means to improve the cell/tissue structural organization

Question 5

Design consideration for bioreactors

Answer 5

1. Definition of Stimuli: Most important
   (stretch, electrical stimulation, perfusion, growth factors)
2. Control of Cultural Variables: temp & pH
   (temp, pH, oxygen saturation)
3. Sensor Technology: latest developed technologies in the field
   (temp, pH, oxygen saturation)
4. Stimulation Protocol: Stretch protocol, etc.
   (feasibility testing, maturation & development, maintenance and sustainability)

Question 6

Scale up vs. Scale out

Answer 6

reduces the operator-dependent variable and ensures product quality
vs.
going from one to multiple

Question 7

Benefits of controlled release of bioactive molecules in vivo

Answer 7

1. Avoids systemic administration
2. Prevent Protease Degradation
3. Sustain Drug Release

Question 8

Physical entrapment, hydrophobic microparticles, non-specific affinity, specific affinity, and covalent attachment

Answer 8

PE: lots of blue strings and red dots
HM: big green dot with red dot inside and some blue strings
NSA: positive and negative charges positive on red dot
SA: blue wings with red dots
CA: black rods connected to red dots

Question 9

Slide 13: Figure 5

Answer 9

Fibrin matrices to used to fuse the carpal joint complex

Question 10

Slide 17: Figure 8

Answer 10

Release of signal 1: low affinity for the matrix
Release of signal 2: intermediate affinity for the matrix
Release of signal 3: high affinity for the matrix

Question 11

What are the three main approaches for 3D bioprinting?

Answer 11

See Slide 4
Biomimicry: This can be achieved by reproducing specific cellular functional
components of tissues, for example, mimicking the branching patterns of the
vascular tree.

Autonomous self-assembly: It requires an intimate knowledge of the
developmental mechanisms of embryonic tissue genesis and organogenesis
as well as the ability to manipulate the environment to drive embryonic
mechanisms in bioprinted tissues.

Mini-tissues: Organs and tissues comprise smaller, functional building
blocks. Mini-tissues can be fabricated and assembled into the larger construct
by rational design, self-assembly, or a combination of both.
3D bioprinting approaches

Question 12

What is the typical process for bioprinting?

Answer 12

See Slide 5
1. Imaging (X-ray, CT, or MRI)
2. Design Approach (Biomimicry, Autonomous self-assembly, or Mini-tissues)
3. Material Selection (Synthetic Polymers, natural polymers, or ECM)
4. Cell Selection (Differentiated cells, pluripotent stem cells, or multipotent stem cells)
5. Bioprinting (inkjet, microextrusion, or Laser-assisted)
5. Application (Maturation, Implantation, and In vitro testing)

Question 13

What are the advantages and disadvantages of extrusion bioprinting?

Answer 13

See slide 7
Advantages
1. Simple process
2. Print large scale construct
3. Selection of various materials
4. High cell viability

Disadvantages
1. Low printing resolution
2. Reproducibility

Question 14

What are the different types of Extrusion-based Bioprinting systems?

Answer 14

See slide 9
1. Pneumatic micro-extrusion
2. Mechanical micro-extrusion
3. solenoid micro-extrusion

Question 15

What are the ideal material properties for bioprinting?

Answer 15

See slide 11
1. Printability
2. Biocompatibility
3. Degradation kinetics and byproducts;
–Degradation rates should be matched to the ability of the cells to produce their own ECM; degradation byproducts should be non-toxic.
4. Structural and mechanical properties
5. Material biomimicry

Question 16

What are the three types of bioink?

Answer 16

See slide 12
1. Hydrogel bioinks (natural and synthetic)
2. Cell aggregate/pellet-based bioinks
3. A combination of the two as hybrid materials

Question 17

What is the process of 3D-bioprinting of cell-encapsulated constructs by using silk-gelatin as a bioink?

Answer 17

See Figure 4 on slide 16
1. Degumming and solubilization of silk in silk fibroin solution
2. Addition of gelatin to make silk/gelatin solution.
3. Incorporate tyrosinase or sonication
4. Add cells
5. Fabrication of 3D cell-laden structure
6. In Situ crosslinking by Tyrosinase or Sonication

Question 18

Microcontact Printing

Answer 18

Reference Figure 2

Question 19

What is Tissue Fusion?

Answer 19

Microtissues adhere to each other and the borders of the microtissues gradually merge to form a large piece of tissue.

Question 20

Micromolding

Answer 20

Reference Figure 3

Question 21

What are the two methods of templated molding of microtissues?

Answer 21

Sacrificial printing and the Kenzan method

Question 22

Describe the Sacrificial Printing Method

Answer 22

Deposits a sacrificial material (agarose or Pluronic F-127) before microtissues are deposited. After tissue fusion the sacrificial material is removed. (Reference Figure 5)

Question 23

Describe the Kenzan method

Answer 23

Point-shaped microtissues are picked and plugged onto the Kenzan using a pick and place robot. Then tissue fusion occurs. Since the Kenzan helps hold the microtissue in position, a 3D tissue construct with the desired shape can be fabricated. (Reference Figure 5)

Question 24

What is the methodology for Cardioid formation?

Answer 24

Tissue culture plates are coated with PDMS and then with natural mouse laminin (a major ECM component). Silk sutures are soaked in highly concentrated laminin and pinned to the center of the plates. Primary neonatal cardiac myocytes are isolated and plated at the confluence on the culture surface. The cell monolayer will delaminate due to the spontaneous contractions of the cardiac myocytes. The monolayer will move to the center of the plates and wrap on the anchor points. Subsequent remodeling results in cardioid formation.

Question 25

What is poly(N-isopropylacrylamide) (PIPAAm)?

Answer 25

A temperature-sensitive molecule

Question 26

How is PIPAAm used?

Answer 26

A cell sheet is laid on a PIPAAm surface at 37C cells adhere to the dehydrated layer. At 20C cells detach from the hydrated PIPAAm forming an intact cell sheet. The change in temp changes the hydrophilicity and promotes H-bonding that repels the cells to create the detached sheet.

Question 27

Describe the conventional approach of guiding self-organization

Answer 27

Identify a mechanism of “animal” organogenesis in vivo. Then by deducing culture conditions for “human” organoid-genesis in vitro from hPSCs.

Question 28

What are the 3 processes of multicellular self-organization?

Answer 28

1. Self-assembly or Sorting Out
2. Self-patterning
3. Self-morphogenesis

Question 29

What are the two advantages of organ-on-a-chip devices?

Answer 29

1. Provide physiologically reasonable microenvironments
2. Provide spatiotemporal controlled microenvironments

Question 30

What is the working mechanism of Hoechst 33342?

Answer 30

Hoechst 33342 binds preferentially to adenine-thymine (A-T) regions of DNA. This stain
binds into the minor groove of DNA and exhibits distinct blue fluorescence emission
spectra that are dependent on dye:base pair ratios.

Question 31

Explain the MSOE bioink and bioprinting process and mechanism.

Answer 31

The bioink used is a novel Pectin/Pluroinc F127 bioink developed at MSOE. Heat-induced gelation of Pluronic F-127 only occurred in the first few
layers of the printed scaffold; however, once the scaffold is removed from the heat, the scaffold
loses its defined shape, becoming a viscous liquid rather than a solid gel. Hence, once the first
few layers are printed, the rest of the scaffold can form a stable gel (not temperature-dependent)
by introducing Ca 2+.

Question 32

Solid-state interaction confer effects

Answer 32

Physical distortion: results from force transmissions between cell and materials due to spatial proximity

Chemical signaling: results in activation/repression of signaling cascades through interactions with insoluble structures

Adhesion points: at subcellular sites at the cell-material interface are physiochemical connections between specialized subcellular sites at the cell membrane and specific components of the material surface

Question 33

Surface chemistry

Answer 33

the chemical properties of the surface of the material in addition to any other modifications that are added to the surface
1.) hydrophobicity v.s. hydrophilicity
2.) Presentation of chemical groups
3.) Patterning of chemical groups

Question 34

Major cell-matrix adhesion receptors

Answer 34

Integrins: are transmembrane heterodimeric proteins that act as mechanotransducers - translate extracellular mechanical stimulations into intracellular responses

Question 35

Mechanotransduction leads to…

Answer 35

changes in gene expression
(Look at Module 8 Figure 19)

Question 36

Cells prefer to migrate from…

Answer 36

softer to stiffer regions

Question 37

Substrate stiffness can influence…

Answer 37

stem cell differentiation along a particular lineage

Question 38

What is surface topography?

Answer 38

physical patterning (architecture) of the material surface

Question 39

Key parameters for developing a polymer microarray

Answer 39

1.) Base substrate
2.) Substrate coating
3.) polymer library
4.) Biological assay

Question 40

Requirements for TE scaffolds

Answer 40

1.) Provide sufficient initial mechanical strength.
2.) Maintains sufficient structural integrity
3.) Possess surface properties
4.) Manufactured under cGMP conditions.
5.) Manufactured economic cost and speed

Question 41

Porosity

Answer 41

A collection of pores, high porosity may provide a greater pore volume for infiltration and ECM formation, but conversely decreases mechanical properties.

Question 42

Interconnectivity and Accessibility

Answer 42

The size of interconnections between pores should be suitably large to support cell migration and proliferation in the initial stages and consequent ECM infiltration of desired tissue and, therefore, is more critical than pore size.
(Look at Module 10 Figure 2)

Question 43

Two-photon Polymerization

Answer 43

The method is a subset of SLA. The resolutions are exceptional with nanoscale tolerances and the ability to produce very small pores and highly detailed structures.
Reactive species generated during 2PP, cause of cellular toxicity

Question 44

Hybrid Fabrication

Answer 44

A common trend is to combine manufacturing approaches so that the benefits of each are provided while minimizing their disadvantages.
Example: electrospinning can be combined with melt electrowriting (MEW) to provide the volume and mechanical properties.
(Look at Module 10 Figure 7)