Exam 1

What is oxidative phosphorylation (respiration)?

Electrons from NAD and FADH2 are used to power the formation of ATP.

When oxygen level is low, what is the preferred substrate between glucose and glutamine?

Glucose is preferred.

How does RGD-containing oligopeptide improve the scaffold's biocompatibility?

RGD - Arg-Gly-Asp • The Arg-Gly-Asp sequence resides in the cell attachment region of fibronectin. • Arg-Gly-Asp-containing peptides support fibroblast attachment, inhibit fibroblast adhesion to fibronectin, and inhibit fibronectin binding to thrombin-stimulated platelets. • In view of the similarities between the binding of fibronectin, fibrinogen, and von Willebrand factor to stimulated platelets, we have examined the effects of Arg-Gly-Asp-containing peptides on the interaction of these latter two adhesive proteins with platelets. ▫ Gly-Arg-Gly-Asp-Ser-Pro used as a prototype peptide inhibited fibrinogen binding to ADP and thrombin-stimulated platelets in the 10-200 µ M range. • The spatial distribution of cell adhesion molecules at the nanometer scale can be controlled using a gel matrix formed from cross-linked alginate molecules containing covalently bound synthetic oligopeptides containing the Arg-Gly-Asp (RGD) sequence (RGD peptides) which mediate cell adhesion Osteoblasts preferentially adhere to specific amino acid sequences such as arginine-glycine-aspartic acid (RGD) and heparin-sulfate binding regions in adsorbed proteins

What is GLUT1?

A primary glucose transporter in most mammalian tissues

Why do you need serial dilution?

A serial dilution is necessary to reduce concentration of cells and reagents working with cells to avoid reaching confluency.

Describe strengths and weaknesses of synthetic scaffolds over natural scaffolds.

Advantages ▫ provide a means to control the initial shape and geometry of the tissue, ▫ provide mechanical support with precise properties ▫ control cell attachment precisely growth factors and other bioactive compounds can be delivered to the cells by means of these scaffold either directly or through their degradation • Disadvantages ▫ challenges in design & matching the amount of ECM. Skeletal tissue has generally less than 2% ECM whereas tendon, ligament, and bone have 30-90% ECM. ▫ presence of any long term, stable, ECM would likely interfere with the normal organization and function of the tissue. This could impact the distribution of forces within the muscle making it difficult for the muscle to contract. Want the synthetic structure to degrade and thus permit intercalation by newly developed ECM.

What is the waste from glycolysis? What is the waste from glutamine metabolism?

Ammonia is the waste from glutamine metabolism and consumption and lactate is the waste from glycolysis

With low glutamine feeding (< 0.2 mM), does the percentile glucose consumption increase, decrease, or remain the same? How about ammonia production? Repeat the question with high glutamine feeding (> 0.2 mM).

Ammonia production increases with higher glutamine feeding and uptake of glucose increases with glutamine consumption

Anchorage dependent (epithelial cells, fibroblasts) vs. anchorage independent cells (macrophages, monocytes, neutrophils).

Anchorage dependent behaves like their tissue of origin and is limited by available surface area. Anchorage independent behaves like a different, amorphous cell.

Describe strengths and weaknesses of biodegradable scaffolds (e.g. PLGA).

Biodegradable scaffolds • Allow the scaffold to be re-adsorbed, gradually being replaced by the cells and the ECM that they produce. • Form a basic support structure • Seed the material with cells • Cells populate the material • Polymer etched or degraded away • Artificial ear • Knee cartilage

How is a biosafety cabinet different from a chemical hood?

Biosafety cabinets are used for protecting the lab and personnel while a chemical hood is used for experimentation

What is cell passaging?

Cell passaging enables an individual to keep cells alive and growing under cultured conditions for extended periods of time.

Why has TE cartilage minimum inflammatory/immune responses? What is the real issue of TE cartilage?

Cartilage consists of primarily extracellular matrix in which chondrocytes are dispersed at a low density within a collagen matrix. • It has no blood supply, nerves, or lymphatics but must be able to resist compression in a dynamic manner. • TE cartilage in theory is a simple development particularly since the chondrocytes can readily be grown in culture. • The primary challenge lies in mimicking the dynamic load normally applied during development.

What is cell immortalization? How can it be done?

Cell immortalization involves an extended replicative capacity which can be achieved by expression of key proteins such as telomerase

What is the function of collagen in ECM? How is it different from elastin?

Collagen is one of the primary constituents of the extracellular matrix • Produced by fibroblasts and osteoblasts (bone cells). • Maintains a complex shape Collagen give strength and structure elastin gives it memory and is primarily produced in fetal period

Describe how the cell density (X) can be evaluated using a spectrophotometer. Calculate the cell density from OD data. Repeat the above question with plate culture. What is colony forming unit (CFU)?

Colonies countedVolume dispersed *10x1x=# of tenfold dilutions

How do you do plating?

Dispense volume of solution on to dish spread onto agar and incubate. Flip upside down so condensate doest get cells.

What is the function of fibronectin in ECM? How is it different from laminin?

Fibronectin sometimes serves as a general cell adhesion molecule by anchoring cells to collagen or proteoglycan substrates. FN also can serve to organize cellular interaction with the ECM by binding to different components of the extracellular matrix and to membrane-bound FN receptors on cell surfacesThe main difference between fibronectin and laminin is that the fibronectin is a glycoprotein found in the extracellular matrix (ECM) and in the blood plasma whereas laminin is a glycoprotein, which serves as the main component of the basal lamina.

Why fluorescence microscope images are originally black-and-white?

Flourescent microscope images are originally black and white to pick up highest contrast and get any light there is.

Describe how fluorescence microscope works.

Fluorescent microscopes work by passing white light through a filter to filter out all but one wavelength that the fluorescent dye emmits to only image one thing.

Describe how you stain focal adhesion sites.

Focal Adhesion sites are stained with FITC

When is the glutamine metabolism affected by glucose concentration? When is the glycolysis affected by glutamine concentration?

For glucose, above a minimum of 1 mM (limiting amount), uptake of glutamine will increase with glutamine concentration • Above this, glucose concentration has no impact on glutamine uptake Below this, glutamine uptake increases greatly, thus reducing lactate production • For glutamine, above a minimum of 0.2 mM (limiting amount), uptake of glucose will increase with glucose oncentration • Above this, glutamine concentration has no impact on glucose uptake • Below this, glucose uptake increases greatly, thus reducing ammonia production

Is glycolysis aerobic or anaerobic? Is TCA cycle aerobic or anaerobic?

Glyclysis is Anaerobic and the TCA cycle is also anaerobic

What is deregulated metabolism?

High and inefficient consumption of carbon, nitrogen, and energy sources leading to increased production of wastes

Increasing the surface roughness on metal implants improves tissue ingrowth. Why?

Increasing the surface roughness gives more areas for the cell to implant on the surface of the implant.

Graphically illustrate how contact angle can be evaluated for biomaterial surfaces. What information can be obtained through contact angle measurement for biomaterial scaffold?

Information that can be obtained through contact angle measurement for biomaterial surfaces include the roughness of the material that can further promote cell adhesion.

Compare two different incubators for bacterial and mammalian cell culture.

Mammilian cell culture needs CO2 and ph balance.

Describe how you stain cell nuclei.

Nuclei are stained with DAPI.

What is photobleaching? How can you prevent it?

Photobleaching occurs when the uv light washes the dye and denatures it making it appear dim and washed.

Injecting β-islet cells from a well-matched donor to a diabetic patients provides only a temporary relief. Why? How can you resolve this issue?

Rejection of the cells A pancreas from a cadaver can be used as an allogenic source of islets of Langerhans, which contain the insulin secreting b-islet cells. • These cells can be injected into the liver of a patient as a graft, but this would normally be rejected providing only a temporary relief. • The longevity of the graft can be increased by administering immunosuppressants or by using tissue from a well-matched donor. •Another way to overcome rejection is by isolating the transplanted cells behind a semi-permeable membrane. •The membrane should permit passage of small molecules such as glucose and insulin, but prevent leakage of cells into or out of the graft.

What is focal adhesion? To identify focal adhesion, what protein molecule is typically imaged? Graphically illustrate all molecules used for such fluorescent staining.

Staint he vincilin to image focal adhesion.

How is Testskin II made?

The Apligraf® technology is comprised of a contracted collagen gel matrix and living keratinocytes and fibroblast cells. The collagen is combined with the fibroblasts to first form a dermal matrix, onto which keratinocytes are seeded to form an epidermal layer. Thus Apligraf® is comprised of the two primary layers of skin.

Why focal adhesion is stronger than non-specific adhesion?

The Specific integrin binding of focal adhesion makes for more traction force on the suface vs non-specific adhesion.

Describe how stains cytoskeleton (actins).

The cytoskeleton actins get stained by TRITC

Why do you need bone marrow transplantation after chemotherapy? What is the best way to harvest bone marrow?

The prolific nature of the bone marrow makes it susceptible to damage from chemotherapy or radiation which impacts rapidly replicating cell types most through damage to DNA. This is why these methods are effective in treating cancers. • Without hematopoetic support a patent would soon need a blood transfusion or would succumb due to hematopoetic failure. • Bone marrow transplant was developed to overcome this problem. Bone marrow is harvested from a patient before chemotherapy. The cells are re-implanted after a few half lifes of the chemotherapeutic treatment has passed.

What property is measured from contact angle analysis? Why is it important for tissue engineering?

The surface roughness is measured from the contact angle analysis and is important to find the surface roughness to determine the hydrophobicity

Describe the typical cell growth curve.

The typical cell growth curve is a logarithmic curve.

Why is a CO2 incubator needed for mammalian cell culture?

They maintain an appropriate pH, 5% CO2, 95% RH, 37 C

How do you do passaging?

To passage cells you must detach them from the surface with trypsine EDTA and take cells to new plate with fresh agar then incubate.

Discuss strengths and weaknesses of using serum as culture medium.

Using Serum can make cells grow fast by introducing growth factors and other low concentration compounds but also disadvantages including high cost, problems with standardization, specificity, variability, and unwanted effects such as stimulation or inhibition of growth and/or cellular function on certain cell cultures

What is focal adhesion? What protein is used in identifying focal adhesion?

Vinculin is the protein located at focal adhesions. Focal adhesion is a type of adhesive contact between the cell and extracellular matrix through the interaction of the transmembrane proteins integrins with their extracellular ligands, and intracellular multiprotein.

With low glucose feeding (< 1 mM), do the percentile glutamine consumption increase, decrease, or remain the same? How about lactate production? Repeat the question with high glucose feeding (> 1 mM).

With low glucose feeding most of the glucose consumed is converted and lactate production is low. In higher glucose feeding most of the glucose feeding the percentage of glucose consumed is low and lactate production is high

What is decellularization? How is it used for tissue engineering applications?

is the process used in biomedical engineering to isolate the extracellular matrix (ECM) of a tissue from its inhabiting cells, leaving an ECM scaffold of the original tissue, which can be used in artificial organ and tissue regeneration. Hearts were de-cellularized by washing with detergent which preserved the underlying extracellular matrix and intact chamber geometry.

Identify lamellipodia and filopodia from the image of cell adhesion.

movement of a motile cell or organism, or part of one, in a direction corresponding to a gradient of increasing or decreasing concentration of a particular substance.Effect of chemoattractants is elicited via chemotaxis receptors, the chemoattractant moiety of a ligand is target cell specific and concentration dependent. • Most frequently investigated chemoattractants are formyl peptides and chemokines. • Chemorepellents are substances expressing adverse migratory effect.

What is chemotaxis? How is it different from molecular diffusion?

movement of a motile cell or organism, or part of one, in a direction corresponding to a gradient of increasing or decreasing concentration of a particular substance.Effect of chemoattractants is elicited via chemotaxis receptors, the chemoattractant moiety of a ligand is target cell specific and concentration dependent. • Most frequently investigated chemoattractants are formyl peptides and chemokines. • Chemorepellents are substances expressing adverse migratory effect.

Define specific growth rate (in equation).

μ = μmax x (S/(Ks x S)). Maximum specific growth rate μmax in time-1, concentration of substrate in solution S in mass/unit volume & half velocity constant Ks in mass/unit volume are the key elements of this calculation.