fModule 3, Day 2: Initiate Cell Culture

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Introduction

Last time you proposed culture conditions for an investigation of chondrocyte phenotype induction or maintenance, and today you will initiate said cultures. The chondrocyte cells you are using were freshly derived from bovine cartilage by enzymatic digestion and immediately frozen. The stem cells were grown from bovine bone marrow over the course of a few weeks, then frozen after 1-2 passages. Cells from cows are often used in part because of their availability from abattoirs. In general, large animals are more useful for modeling human joint diseases such as osteoarthritis than are small animals, because the resting angle of their knee joints is more similar to that of humans. In this module, we will work with an in vitro culture model of cartilage-forming cells.

Your two cell samples will be grown in alginate bead cultures. You have probably encountered alginates many times in your life, as thickeners in food and textiles, preservatives, and possibily at your dentist or in a pharmacy. Alginate is a polysacharride derived from seaweed, a co-polymer of mannuronic and guluronic acid. A single alginate molecule may contain long stretches of either acid (called M-blocks and G-blocks), as well as random and/or strictly alternating G/M sequences. The precise chemical composition of an alginate determines its mechanical properties, degradability, and other important characteristics. Qualities such as strength and viscosity are also influenced by the average length of the individual polymer chains (i.e., the molecular weight), and by alginate concentration. For example, high molecular weights correlate with increased viscosity. Alginates in general are shear-thinning, which is to say their viscosity decreases as shear rate increases (e.g., when quickly drawn into a syringe).

Schematic of cross-linked alginate. G-blocks are represented by dotted lines, M-blocks by curved solid lines, and calcium ions by green circles.

Cations such as calcium can cross-link alginate chains to form a network, or gel. The identity and concentration of the cross-linker influence the ultimate material properties. Only G-blocks can be linked to each other, while M- or MG-blocks cannot, but in turn provide flexibility (see figure). The resultant semi-solid structure has the capacity to hold a large amount of water, and the water-swollen structure is called a hydrogel. Hydrogels have several attractive properties for tissue engineering: they allow oxygen and nutrients to diffuse better than non-hydrated materials do; their mechanical and biochemical properties are readily varied by co-polymerization of multiple elements; they mimic the elasticity of natural tissues, and they often form rapidly and under mild conditions. Some gels can be injected into a patient in liquid form, then solidified within his or her body by heat or light. Such injectable gels have the advantage of easily filling an arbitrarily sized wound shape, which is difficult for implantable gels to do. Natural (e.g., alginate) and synthetic (e.g., poly(ethylene glycol)) hydrogels each have distinct advantages and disadvantages, as we will discuss in class.

Today you will make alginate hydrogels in bead form, by slowly releasing alginate solution from a syringe into a bath containing calcium chloride. Next time you will see how well your cells survived.

Protocols

Half the class at a time will work in the tissue culture room today. The other half of you will explore the NCBI bovine information site, and otherwise spend the time however you find useful ("For Next Time" assignment on design plan and expected assay results, notebook prep, or unrelated work).

Part 1: Chondrocyte or Stem Cell Culture

Today you will work with primary cells that are directly isolated from bovine knee joints. Recently, your teaching faculty harvested cartilage fragments from two bovine knees, and sequentially digested them in pronase and collagenase enzymes. Each joint typically yields > 50-100M cells. After cell isolation, aliquots of several million cells each were frozen and stored in liquid nitrogen.

Preparation

1. Begin by setting up your hoods. Prepare any standard equipment and solutions needed.
2. Note that the small beakers are for making a calcium chloride bath (not shared, one per person), and the large are for temporary waste in steps 10-12 below (shared, one per hood).
3. If you requested a special reagent or equipment, check with the teaching faculty.
4. If you are doing an alternative protocol (e.g., 2D culture or collagen gels), check with the teaching faculty.

Cell culture

1. When your hood is ready, thaw your aliquot(s) of frozen cells in the water bath. Avoid immersing the cap of the tube in the bath, just hold the body submerged. Agitate the vial slightly while you hold it. The cells should thaw in less than 5 minutes.
2. Spray the vial with 70% ethanol and take it into your hood. Using a P1000, add the cells drop-wise into the 15 mL conical containing 9 mL of pre-warmed medium. Spin at 800 g for 8 minutes.
3. Aspirate most of the medium off your cell pellet, then gently resuspend in 1 mL of medium using your P1000. Add 3 mL more of medium per vial, using a serological pipet for the addition and subsequent mixing of the medium and cells. Take 90 μL of cells into an eppendorf tube.
4. Add 10 μL of Trypan blue - this is a toxic material, so please be careful not to spill it! - to the eppendorf tube, and count your cells. Adjust your culture plan if you do not have as many cells as you expected.
   • No need to count all 4 corners today - perhaps count 2, especially if your cell count is high.
5. Separate the cells that will make up your two different cultures into two labeled 15 mL conical tubes. Note that the tubes may not all require the same amount of cells, depending on the cell densities you chose for the two cultures. Double-checking your calculations now may save you having to do an extra centrifugation step later!
   • Give any excess cells that you have to the teaching faculty, in case other groups want more cells.
6. Spin down your two conical tubes of cells at 800 g for 8 minutes.
7. Resuspend each sample of cells in the appropriate amount of the type and concentration of alginate that you chose.
8. Using the syringe that has been prepared for you, very carefully pull up the cells, then release them drop-by-drop into the beaker full of calcium chloride solution (20 mL). Recall that calcium effectively polymerizes the alginate, resulting in small gel beads filled with cells. Immediately discard the entire syringe into the sharps container - do not try to remove or recap the needle.
   • Don't release too quickly or you will get a glob instead of distinct droplets, and try to match your release rate with your partner's.
   • Depending on the concentration of alginate that you chose, you may have between ~50-150 beads for 1 mL of alginate solution.
9. Allow the polymerization to proceed for 10 min. at room temperature. Then pour your beads into a 50mL conical tube.
10. Remove the calcium chloride solution from your beads using a large serological pipet (to better avoid aspirating the beads), and put this solution in the temporary waste beaker in your hood.
    • Ask the teaching faculty for tips on avoiding sucking up your beads. Basically, you want to keep the pipet close to the wall of the conical tube, so liquid can still be sucked up but the beads don't have room to be.
11. Now fill the conical tube with sodium chloride (20 mL), and gently shake it for 1-2 min. This is to remove excess calcium from the solution.
12. Remove the NaCl using a fresh pipet, then wash the beads again with fresh NaCl. Finally, wash the beads two times with DMEM culture medium (20 mL each time).
13. For each of your two samples, transfer the beads to the two leftmost wells of a 6-well plate, using a sterile spatula. Try to put approximately equal numbers of beads in the two wells.
14. Finally, add 6 mL of warm culture medium to your four sample wells, then put the two well-plates in the incubator.

The teaching faculty will exchange the culture medium as necessary.

Part 2: Primers for RT-PCR

Did you know that NCBI has a Web site devoted to all things cow? NCBI Bovine Genome Resources

It's true! And today you will use this site to find the primers you need to perform RT-PCR on Day 4 of this module. Try searching for collagen types I and II (the alpha chain of each is fine) in the Map Viewer (upper right of page). What chromosome is each collagen chain located on? See if you can make your way to the UniSTS entries for collagen, which list recommended primers for RT-PCR. How long are the expected PCR products if these primers are used?

Another option for finding primer suggestions is looking in the literature. Of course, this can be a risky proposition, but if you verify the primers against information in the NCBI database, it can be faster than making your own from scratch, and provide a feeling of security (someone, somewhere has succesfully amplified the sequence in question!). The paper by Ikenooue, et al. lists primers recommended for collagen type II. What species are the primers for? If it's not bovine, you cannot use the primers directly. However, you can BLAST the primers against the bovine genome, similar to what you did in Module 2 to verify your mutagenized plasmids against the original, or Module 1 to search for homology in your RNA aptamers.

Go to the BLAST Web site and select the bos taurus genome. Type in the primers from the journal article one at a time, then perform the BLAST as follows: select BLASTN, change the "Expect" value to 0.1, and turn off the low complexity filter. How many nucleotides changed between the human and cow for each primer?

Why must you use cDNA rather than complete genes (introns+exons) when making primers for RT-PCR?

References:

• Ikenoue, T., et al. "Mechanoregulation of Human Articular Chondrocyte Aggrecan and Type II Collagen Expression by Intermittent Hydrostatic Pressure in vitro." J Orthop Res 21, no. 1 (Janurary, 2003): 110-6.

Design Plan and Expected Assay Results

On Day 1, one of the For Next Time assignments was to prepare a brief description of your design plan and expected assay results by the end of Day 2's lab session. 

Following are two examples posted by students.

T/R Pink (Ariana Chehrazi and Jacqueline Söegaard)

• Plan: Since mechanical loading and pressure in the joints is one of the factors that contributes to cartilage degradation in secondary osteoarthritis, through our experimental design, we seek to evaluate the effect of compression and pressure on the ability to grow a 3D chondrocyte culture. We will grow two cultures in a six well plate: both cultures will be covered with a square glass slide (m = 0.189 g, A=22 mm x 22 mm), and one of them will also be subjected to additional pressure by placement of a metal mass (m= 24.608 g) on top of the glass slide. (Cell culture conditions: Sigma Aldrich "low viscosity" alginate at 1.5%; Differentiated chondrocytes, 10^7 cells/mL, media conditions are standard).
• Expectations: We think that the control (weightless) sample will preserve a chondrocyte-like phenotype, whereas the compressed sample will lose chondrocyte phenotype and will possible not be as viable, since we think compression and confinement in the scaffold will contribute to chondrocyte degradation.

W/F Red (two anonymous MIT students)

• Plan: Make beads in different concentrations of CaCl2: 51 mM and 204 mM, so that the alginate will have different degree of cross linking and thus different stiffness.
  • Cell type: chondrocytes
  • Cell density: 5 million cells/ mL
  • Total # cells needed: 10 million
  • Alginate: Protanal LF120M
• Expectations: A paper by Gene et al reported that the mechanical properties of the scaffold affect the cell phenotype. The stiffer the alginate is, the more they found fibroblast phenotype. Ca2+ will cause cross linking of the alginate so we expect that the higher [CaCl2] (204 mM), the stiffer the alginate will be, and the more fibroblast phenotype will be found compared to the 51 mM. We expect that more fibroblast phenotype will correspond to a higher Collagen II expression (compared to Collagen I).

For Next Time

1. Sign up for a time to do your Day 3 lab work; on this day you will arrive at staggered times.
2. For each well in the six-well dish you and partner seeded on Day 1, calculate the number of cells you expect to have after 120 hours. Show all your work, starting from the raw hemocytometer data. The following rules of thumb and guesses should be used for your calculation, and you should provide two final answers, one for each dilution:
   • only 25% of the cells are able to stick and proliferate (this is called a 25% plating efficiency).
   • the doubling time for the cells is 24 hours.
   • the cells take 24 hours to recover from trypsin treatment before they begin doubling.
3. The primary assignment for this experimental module will be for you to develop a research proposal and present your idea to the class. For next time, please describe five recent findings that might define an interesting research question. You should hand in a 3-5 sentence description of each topic and cite the reference that led you to each item. The topics you pick can be related to any aspect of the class, i.e. RNA, protein, or cell-biomaterial engineering. During lab next time, you and your partner will review the topics and narrow your choices, identifying one or perhaps two topics for further research.
   • Note: for now, you do not have to have a novel research idea sketched out; you simply have to describe five recent examples of existing work. However, you can start to brainstorm how to build off of those topics into something new if you want to get ahead of the game.

Reagent List

• Media as described on Day 1, or with special additives if requested
• Alginates as described/requested on Day 1
• 102 mM CaCl2
• 0.15 M NaCl
• Trypan blue, 0.4%