Jun Zhang, Hongxu Qi, Hongjun Wang, Ping Hu, Lailiang Ou, Shuhua Guo, Jing Li, Yongzhe Che, Yaoting Yu, Deling Kong (2006) Artificial Organs 30 (12), 898–905.
One of the main problems faced by cardiologists and heart surgeons is the low availability of small diameter (less than 5mm) blood vessels that are used in surgical replacements and bypass surgeries.As a result, there has been an increase in efforts to design small diameter artificial vascular grafts.In previous studies, the long-term patency, or state of being open and unblocked, of tissue engineered blood vessels smaller than 6 mm in diameter has been poor.This paper shows the development and growth of small diameter vessels by seeding genetically modified mesenchymal stem cells (MSCs) onto a polymer scaffold.
Nitric oxide (NO) is an important factor in the regulation of proper blood vessel regulation.Its production also has a strong positive correlation with the patency of blood vessels used in bypass surgery.In this paper it was hypothesized that grafts engineered with endothelial nitric oxide synthase (eNOS)-modified MSCs would produce an NO level comparable to that of native blood vessels, and greater than that of unmodified MSCs.In order to make the small vascular grafts, MSCs were extracted from the bone marrow of rats, expanded in culture, and modified with eNOS.The cultures were then seeded on a tubular poly (propylene carbonate) (PPC) graft which was produced by electrospinning.Grafts of both eNOS-modified and unmodified MSCs were developed, with a rat artery of similar size as a control.
A number of the techniques that we will be using or already have used in our lab classes were performed for this paper.RT-PCR was used to quantify the amount of eNOS expressed in the grafts.Proteins in the grafts were separated and identified using western blotting.Fluorescence microscopy was used to examine the immunohistochemistry of the cultures.The results of the study showed that NO production level of the non-eNOS modified MSC grafts was similar to the baseline, whereas the eNOS-modified MSC grafts produced NO levels comparable to that of the control vessels.
This paper is interesting because it helps show the importance of understanding the regulatory pathways of different tissues and organs when designing artificial organs.They also use a number of techniques that we will be using in lab.Heart and vascular disease/problems are on the list of top killers in the world1.I think it is important to investigate both means of preventing these diseases and means of repair/regeneration (in this case, replacement of artificial blood vessels) upon occurrence of the disease.In future studies, it would be interesting to establish what other regulatory pathways may have been compromised in previous designs of vascular grafts less than 5mm.Additional studies should also be done showing the effects of different mechanical forces on these eNOS-modified MSC grafts, to ensure that they would be able to withstand forces that occur naturally within the vascular system.It would also be good to measure the patency of these grafts compared to grafts made with other polymers in vivo to determine which material has the best long-term outcome under “normal” conditions of the body.
Rhima M. Colemana, b, Natasha D. Casea and Robert E. Guldberg Biomaterials Volume 28, Issue 12, April 2007, Pages 2077-2086
The use of chondrocytes, a type of cells that secretes cartilage matrix, for cartilage tissue engineering has presented several challenges. It is difficult to harvest enough number of chondrocytes without dedifferentiation. Passaging chondrocytes usually results in decreased production of desirable cartilage matrix components such as of type II collagen, sulfated glycosaminoglycans (sGAGs), and increased production of undesirable type I collagen. Alternatively, bone marrow stromal cells (BMSCs), which have a similar characteristic to stem cells of being able to differentiate into bone, cartilage, and fat under proper conditions, has been successfully cultured in 3D and shown to possess chondrogenesis. The purpose of this paper is to investigate the effects of biochemical factors (i.e. growth factors) and focus on choices of hydrogels as scaffold materials on chondrogenic differentialtion of rat BMCs.
The main growth factors being investigated in this study include transforming growth factor-β1 (TGF-β1), fibroblastic growth factor-2 (FGF-2), and Dexamethasone (Dex). TGF-β1 enhances differentiation of stromal cells at late passages whereas FGF-2 treatment enhances TGF-β1. While Dex upregulates the production of TGF-β1 mediated collagen II and is required for chondrogenesis, it is also shown to have negative effects on cells viability. Initial data from this study showed that the treatment of FGF-2 during monolayer expansion and TGF-β1 in the presence of Dex during 3D culture in hydrogel culture of P2 yielded the greatest production of sGAG, a cartilaginous matrix. This optimal condition was then used to study the effect of hydrogels-alginate and agarose. Although 2% alginate and agarose used in this study have similar macroscopic property such as pore size, they were shown to have different effects on cell behaviors in response to biochemical stimulations. In alginate, the presence of FGF-2 enhances the effect of TGF-β1 and results in more production of a cartilaginous matrix than the presence of TGT- β1 alone. The opposite result was found when BMSCs were cultured in agarose. Moreover, Dex did not show any negative effect on cell death when being used in alginate cultures.
The techniques used in this study were mostly the same techniques we have learned in BE115 class such as cell passaging, 3D cell culturing, cell lysis, live/dead assays. Measuring cell’s production of excretion and cartilaginous protein was a good example of cell characterization based on cell’s responses to different stimuli and environments. Live/dead assay using green and red dyes as molecular probes with a confocal microscope was used to determine cell death in gel samples. The use of software such as ImagePro, in this case, to count the number of cells in live/dead assay was proved to be necessary and useful.
Wednesday, February 21, 2007
Lab 7: SDS-PAGE
1 Introduction
Protein electrophoresis is an extremely popular technique in molecular biology. Simply, proteins (typically from a cell or tissue lysate) in an SDS-containing buffer are added to the top of a polyacrylamide gel. The SDS, a powerful anionic surfactant, serves to surround the protein, overwhelming its inherent charge. The protein surrounded by the negatively-charged SDS has a net negative charge approximately proportional to its mass. When a potential difference is applied across the gel, the negatively charged proteins migrate through it. Smaller proteins migrate more quickly through the gel, and the proteins are separated by size into ‘bands’.
Sodium Dodecyl Sulfide – PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is commonly followed by either total protein staining or transfer and Western blotting.
2 Objectives
- To separate proteins in a lysate by molecular weight. - To prepare gels for total protein staining or Western blotting.
5. Resolving gel materials (amounts for 2-3 6% acrylamide gels)
The resolving gel is the gel that is poured first; in it, proteins are resolved into discrete bands.
8.0 mL dH2O 3.0 mL Acrylamide/Bis-Acrylamide 3.75 mL 1.5 M tris-HCl, pH 8.8 150 µL 10% (w/v) SDS 75 µL 10% (w/v) Ammonium persulfate (APS) in dH2O, made fresh on day of use 10 µL TEMED
6. Stacking gel materials (amounts given for 2-3 4% acrylamide gels)
The stacking gel is poured on top of the resolving gel after it has finished gelling, with a ‘comb’. Protein samples are added to the individual wells formed by the stacking gel gelling around the comb.
3.0 mL dH2O 666 µL Acrylamide/Bis-Acrylamide 1.25 mL 0.5 M tris-HCl, pH 6.8 50 µL 10% (w/v) SDS 5 µL 1% (w/v) Bromphenol blue 25 µL 10% (w/v) Ammonium persulfate (APS) in dH20, made fresh on day of use 5 µL TEMED
7. 5X gel-running buffer
This buffer is diluted to 1X with dH2O to make gel running buffer. Gel running buffer is used to fill the electrophoresis cell (or bath), keeping the gel wet and allowing the electrophoresis unit to operate.
(5x)125 mM Tris base (30.3 g) (5x)0.960 M glycine (144 g) (5x)0.1% SDS (10 g)
Add the quantities in parenthesis above to dH2O until total volume is 1800 mL, bring to pH 8.3, then add more dH2O for final volume of 2000 mL. Store at room temperature and dilute to 1X with dH2O before use.
8. 2X SDS-PAGE sample buffer
This is the buffer which protein samples are diluted with before being loaded into the gel for analysis.
9. Protein samples
10. Transfer buffer - Do NOT pH, make fresh on the day of use
The transfer buffer is used when transferring proteins from the gel that you poured to a nitrocellulose membrane (for Western blotting).
25 mM Tris base (3.0 g) 0.2 M glycine (15 g) 20% methanol (200 mL)
Add the quantities in parenthesis above and bring up to 1000 mL total volume with dH2O.
11. Tris Buffered Saline with Tween-20 (TBST)
TBST is used to wash the nitrocellulose membrane.
20 mM Tris-HCl (3.14 g) 137 mM NaCl (8.0 g) 0.1% Tween-20 (1 mL)
Add the quantities in parenthesis above to 900 mL dH2O, pH to 7.5, bring to final volume of 1000 mL with dH2O. Store at 4°C.
4.2 Supplies
1. 1.5, 15, and 50 mL centrifuge tubes 2. 10, 100, and 1000 µL pipette tips 3. 5 and 10 mL pipette 4. Nitrocellulose membrane 5. Filter paper
4.3 Equipment
1. Mini-PROTEAN 3 Cell and Systems 2. Power supply
5 Protocol
5.1 Assembling the gel casting unit
1. Cover your lab bench with paper if you have not already done so.
2. Use distilled water to clean all electrophoresis equipment. Wipe with Kimwipe, and set the components out to dry.
3. Assemble casting stand as shown below.
4. Check assembly for leaks using distilled water. Fill the space between the glass plates; if the unit leaks, reassemble and try again. If not, pour the water out and dry between plates using a folded paper towel, Kimwipe, or filter paper.
5. Begin water boiling for later use.
5.2 Pouring the Resolving Gel
1. Mix all resolving gel components together except the 10% APS and TEMED in a 50 mL centrifuge tube. Seal tube and tip back and forth gently to mix.
2. Add 10% APS and TEMED, seal tube and tip back and forth gently to mix..
3. Transfer solution to each of the two plate sandwiches using a 5 or 10 mL pipette. Do not fill all the way! There should be ~1 cm of space between the top of the short plate and the resolving gel level (right in the middle of the green plastic bar behind the glass plates).
It is important that you add the correct amount of gel. Too little and your resolution will be poor; too much, and there won’t be room for a stacking gel on top. Be careful to avoid bubbles, which will inhibit polymerization and distort protein migration. Replace leftover gel in centrifuge tube.
4.Immediately after pouring the resolving gel, using a pipette or wash bottle slowly and very gently add just enough 0.1% SDS solution to cover the resolving gel without disturbing it. The SDS solution is there to keep the gel from drying out and to protect it from oxygen, which will inhibit the reaction.
5. Wait 15-30 minutes. Check to see that the leftover gel in the centrifuge tube has polymerized, and if it has, tilt the gel former slowly to confirm that the resolving gel under the SDS solution has polymerized completely.
Pour the SDS solution into a Kimwipe, and rinse the top of the gel very gently with dH2O.
5.3 Pouring the Stacking Gel
1. Mix all stacking gel components together except the 10% APS and TEMED in a 15 mL centrifuge tube.
2. Add 10% APS and TEMED, seal tube and invert several times to mix. Add stacking gel solution to the top of the resolving gel using 5 mL pipette, returning extra solution to centrifuge tube.
The stacking gel solution should almost fill the remaining space – leave ~ 2-3 mm between the top of the stacking gel and the top of the short plate.
Immediately thereafter, insert the comb. Start from one side and 'brush' air bubbles off to one side.
3. Wait 5-15 minutes. Check to see that leftover gel in the centrifuge tube has polymerized.
5.4 Assembling the electrophoresis unit
1. After polymerization, remove your gel sandwiches gently from the gel casting apparatus and transfer them both into the electrophoresis unit as shown below.
When assembling the electrophoresis unit, the short plates must face inward!
2. Place the electrophoresis unit into the electrophoresis bath. Fill the inner chamber (the space between the two gels) with gel running buffer, and check for leaks.
3. Add approximately 250 mL of gel running buffer to the outer chamber (the clear plastic electrophoresis bath). 250 mL is about 4-5 cm of buffer, measured from the bottom. While the inner chamber must be filled, the outer chamber need not be completely filled.
4. Remove comb carefully and gently rinse each loading section of the gel gently with the gel running buffer using a 1000 μL pipette.
5. Set up equipment near power source.
5.5 Electrophoresis
1. Add 1 part 2X sample buffer to 1 part protein samples and standards.
2. Heat protein samples and standards (do not heat the molecular weight ladder unless the manufacturer suggests it) at 100 C for 5 minutes.
3. Put your samples on ice for ~60 seconds.
4. Centrifuge samples briefly to remove bubble and pellet any undissolved cell extract. Your protein will be in the supernatant; any pellet should be left undisturbed.
5. Using a 100 µL pipet tip, add protein samples to the wells in the gels as shown below. Be very careful! It’s easy to miss a well entirely if you’re not paying attention. Pipette very slowly.
The gel can be overloaded two ways - too much protein, or too much volume. Too much protein will separate badly; I suggest loading no more than ~40 µg of total protein. The maximum volume depends on the type of comb and the spacer plates used. For an 8-well comb using 0.75 mm spacer plates, I suggest using no more than 25 µL sample volume.
6. Run the gels at 100-150 V until the dye front has nearly reached the bottom of the gel (approximately 60 minutes).
5.6 Disassembly and Transfer
1. When finished, disassemble the electrophoresis unit.
2. Prepare a bath of transfer buffer (for a Western blot) and a bath of dH2O (for total protein staining).
3. Use a wedge to gently separate one of the glass plates from the gels.
The gel might tend to stick at the top, where the stacking gel was. It doesn't matter which plate you remove, as long as you can remove one leaving the gel on the other. Be careful! It's easy to tear the gel at this point.
4. Carefully take a folded Kimwipe and place it atop the stacking gel (small gel on top with comb) without touching the resolving gel (larger gel on bottom without comb). The edge of the towel should line up with the separation between resolving and stacking gel. Press the paper towel firmly against the stacking gel, then pull the paper away.
The stacking gel should neatly come with the paper, and can be discarded. If it doesn't all come off at one, try again.
5. Submerge the gel in the bath as shown below. If necessary, use the wedge to loose the gel into the buffer bath.
5.6a Total protein staining
Typically total protein staining is done with Coomassie or silver stain. The protocol varies depending on the reagent that you use. This will be done next week.
5.6b Transfer for Western blotting
1. Cut a piece of nitrocellulose membrane and pieces of filter paper so that their size matches that of the gel. Try to avoid touching the nitrocellulose membrane, manipulating it only at the edges. After your gel has incubated in transfer buffer for ten minutes, add the membrane to the transfer buffer.
2. After your membrane has incubated in transfer buffer for five minutes, dip the filter paper and sponges into the transfer buffer. Using the mini trans-blot unit and the gel, make a sandwich of the following:
RED - sponge - paper - membrane - gel - paper - sponge - BLACK
Do not try to take the gel out of the bath without support. Slip either the membrane or the wedge underneath the gel, using it for support.
Don’t let the gel dry out, and pour a little extra transfer buffer over it if it's getting sticky and you're having trouble manipulating it.
3. Before sealing it, roll a pipette over the top paper layer of the sandwich to ensure that the membrane and the gel are pressed against each other without any air bubbles that will interfere with the transfer.
4. Insert this sandwich into the blotting apparatus and pour in enough transfer buffer to fill the transblot apparatus.
5. Run at ~100 V for ~ 60 minutes or ~25 V overnight in the refrigerator to allow transfer of proteins from the gel to the nitrocellulose.
6. Turn off mini trans-blot unit. Remove mini trans-blot sandwich, and carefully peel nitrocellulose membrane from gel. Wash briefly with TBST. Your membrane is ready to be blocked, probed with antibodies, and developed.
Bone grafts are typically needed when fractures fail to mend, there is substantial bone loss after severe injury, necrosis due to avascularization, or for bone reconstruction post tumor removal (i.e. bone marrow cancer).These cases are usually treated with autogenous bone grafts.As with any implantation/transplantation, donor health (site of treatment), mechanical performance of the implant, vascularization, sterilization, and cost are critical considerations for successful treatment.To ameliorate or even alleviate these issues, the field of tissue engineering has lately been developing ideal bone graft substitutes.
There are three properties to characterize an ideal bone graft substitute: using 1) osteoconductive scaffolds, 2) osteoinductive proteins, that in turn maintain 3) osteogenic cells.Experimental implants were created using a combination of a coral exoskeleton scaffold, with and without bone morphogenic protein (BMP-2) and with or without a vascular pedicle.The bioreactors were harvested in rats.Via histology, actual bone formation (osteocytes, osteoblasts, and osteiod regions) and new vascularization only occurred in the presence of BMP-2 and a vascular pedicle (and the coral scaffold).
This paper was particularly interesting because bone formation can provide a model for the study cancer.It is believed that the same processes that occur in osteogenesis within a scaffold, cell differentiation and proliferation are very similar to that of tumor metastisis.Furthermore we talked about the use of controls and their importance in experimental design.Though seemingly having a minor effect, without the controls constructed in this experiment, key factors of bone formation could not be understood. And in turn tumor metastisis dynamics would have that much less potential for understanding.
This paper describes the use of a 3-dimensional weave of biodegradable fibers that can be infused with a hydrogel (agarose is on the list of hydrogels they use!) containing cells. The woven composite has anisotropic mechanical properties similar to cartilage - more similar, at least, than agarose + cells alone.
Long-term cell survival has not yet been characterized, but short-term survival - including the vacuum-assisted process of squeezing the hydrogel into the cells, it promising. You'll note that they used the same Live/Dead assay as we discussed in lecture 4.
There are a lot of similarities here with our class projects, with the addition of the weave, and the paper demonstrates a few common experiments that we've discussed briefly in class. It also shows where you can take the relatively simple cultures we'll be making this week in lab once you finish with the course.
