Cell Culture- Master Deck Flashcards
Major function of bioreactors:
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
Effects of perfusion on cell seeding
Cells will penetrate the whole scaffold and perfuse more homogenous
Methods for estimating number of cells in a reaction
- Glucose or oxygen consumption
- Specific product: (hepatocytes monitor urea, fibroblasts produce collagen, general: lactate…yeast produces ethanol)
Role of physical conditioning
- 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
- DIFFERENTIATION: Mechanically generated signals play a significant role in differentiation of progenitor cells into different lineages
- ORGANIZATION: Effective means to improve the cell/tissue structural organization
Design consideration for bioreactors
- Definition of Stimuli: Most important
(stretch, electrical stimulation, perfusion, growth factors) - Control of Cultural Variables: temp & pH
(temp, pH, oxygen saturation) - Sensor Technology: latest developed technologies in the field
(temp, pH, oxygen saturation) - Stimulation Protocol: Stretch protocol, etc.
(feasibility testing, maturation & development, maintenance and sustainability)
Scale up vs. Scale out
reduces the operator-dependent variable and ensures product quality
vs.
going from one to multiple
Benefits of controlled release of bioactive molecules in vivo
- Avoids systemic administration
- Prevent Protease Degradation
- Sustain Drug Release
Physical entrapment, hydrophobic microparticles, non-specific affinity, specific affinity, and covalent attachment
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
Slide 13: Figure 5
Fibrin matrices to used to fuse the carpal joint complex
Slide 17: Figure 8
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
What are the three main approaches for 3D bioprinting?
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
What is the typical process for bioprinting?
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)
What are the advantages and disadvantages of extrusion bioprinting?
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
What are the different types of Extrusion-based Bioprinting systems?
See slide 9
1. Pneumatic micro-extrusion
2. Mechanical micro-extrusion
3. solenoid micro-extrusion
What are the ideal material properties for bioprinting?
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
What are the three types of bioink?
See slide 12
1. Hydrogel bioinks (natural and synthetic)
2. Cell aggregate/pellet-based bioinks
3. A combination of the two as hybrid materials
What is the process of 3D-bioprinting of cell-encapsulated constructs by using silk-gelatin as a bioink?
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
Microcontact Printing
Reference Figure 2
What is Tissue Fusion?
Microtissues adhere to each other and the borders of the microtissues gradually merge to form a large piece of tissue.
Micromolding
Reference Figure 3
What are the two methods of templated molding of microtissues?
Sacrificial printing and the Kenzan method
Describe the Sacrificial Printing Method
Deposits a sacrificial material (agarose or Pluronic F-127) before microtissues are deposited. After tissue fusion the sacrificial material is removed. (Reference Figure 5)
Describe the Kenzan method
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)
What is the methodology for Cardioid formation?
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.
What is poly(N-isopropylacrylamide) (PIPAAm)?
A temperature-sensitive molecule
How is PIPAAm used?
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.
Describe the conventional approach of guiding self-organization
Identify a mechanism of “animal” organogenesis in vivo. Then by deducing culture conditions for “human” organoid-genesis in vitro from hPSCs.
What are the 3 processes of multicellular self-organization?
- Self-assembly or Sorting Out
- Self-patterning
- Self-morphogenesis
What are the two advantages of organ-on-a-chip devices?
- Provide physiologically reasonable microenvironments
- Provide spatiotemporal controlled microenvironments
What is the working mechanism of Hoechst 33342?
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.
Explain the MSOE bioink and bioprinting process and mechanism.
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+.
Solid-state interaction confer effects
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
Surface chemistry
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
Major cell-matrix adhesion receptors
Integrins: are transmembrane heterodimeric proteins that act as mechanotransducers - translate extracellular mechanical stimulations into intracellular responses
Mechanotransduction leads to…
changes in gene expression
(Look at Module 8 Figure 19)
Cells prefer to migrate from…
softer to stiffer regions
Substrate stiffness can influence…
stem cell differentiation along a particular lineage
What is surface topography?
physical patterning (architecture) of the material surface
Key parameters for developing a polymer microarray
1.) Base substrate
2.) Substrate coating
3.) polymer library
4.) Biological assay
Requirements for TE scaffolds
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
Porosity
A collection of pores, high porosity may provide a greater pore volume for infiltration and ECM formation, but conversely decreases mechanical properties.
Interconnectivity and Accessibility
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)
Two-photon Polymerization
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
Hybrid Fabrication
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)
