Hippocampal Neuron Dissociation Transfection and Culture in Microfluidics Chambers

Materials and Reagents

1. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
   
2. HBSS (Hyclone, catalog number: SH30268 )
   
3. Boric acid (Sigma-Aldrich, catalog number: B0252 )
   
4. Borax (Sigma-Aldrich, catalog number: B9876 )
   
5. Cysteine (Sigma-Aldrich, catalog number: C7352 )
   
6. EtOH
   
7. Neural basal media (NBM)
   
8. Glutamax
   
9. B27
   
10. PenStrep
    
11. Gentamicin
    
12. Glutamate
    
13. FBS
    
14. Laminin
    
15. Trypan blue
    
16. Na₂SO₄
    
17. K₂SO₄
    
18. MgCl₂
    
19. CaCl₂
    
20. Glucose
    
21. Phenol Red
    
22. EDTA
    
23. 0.1 M borate buffer (pH 8.5) (see Recipes)
    
24. Dissection media (DM) (see Recipes)
    
25. Papain activation buffer (see Recipes)
    
26. Papain (100 mg) (Worthington Biochemical Corp, catalog number: LS003119 ) (see Recipes)
    
27. DNase (F. Hoffmann-La Roche, catalog number: 10104159001 ) (see Recipes)
    
28. Papain digestion media (see Recipes)
    
29. Dissociation media (see Recipes)
    
30. Plating media with phenol red (see Recipes)
    
31. Growth media without phenol red (see Recipes)
    

Equipment

1. PDMS devices
   
2. Microscope
   
3. Hemacytometer
   
4. TC hood
   
5. Incubator
   
6. -80 °C freezer
   
7. 37 °C water bath
   
8. 6 cm cell culture dishs
   
9. Plasma-bonding machine (model PDC-32G , http://www.harrickplasma.com/)
   

Procedure

1. Coverslip and Chamber Preparation
   Day 1:
   1. PDMS devices are fabricated as described in Park et al. (2006).
   Day 2:
   2. Corning No.1, or No.1.5 coverglasses are both suitable for our microscope (24 mm x 40 mm). Or, Carolina coverglasses 24 mm x 50 mm.
      1. Sonicate the cover glasses in autoclaved ddH₂O for 60 min.
         
      2. Sonicate again in 70% EtOH for 30 min.
         
      3. Sonicate in 100% EtOH for 30 min.
         
      4. Wash with autoclaved ddH₂O in TC hood, air dry the coverglasses overnight in the hood.
         
   Day 3:
   3. Assemble the coverglass and PDMS device by using the plasma-bonding machine.
      
   4. Press firmly on the corners but only lightly touch the middle channel. Coat the chamber with PDL (>150K mol wt, prepared in borax boric acid buffer):
      1. 0.5 mg/ml PDL is added to a well on one side of the device and allowed to flow through into the connecting well (leave the other side unfilled).
         
      2. Allow the PDL to flow through the device for about 10 min and then add more PDL to the wells to fill them up.
         
      3. Place the devices containing PDL in an incubator at 37 °C for a minimum of 30 min to let the PDL fill the micro-groves.
         
      4. Fill the wells and major channel on the other side of the chamber with PDL and return the chamber to incubator at 37 °C for a minimum of 4 h, overnight is preferable.
         
   Day 4:
   5. Pipette out the excess PDL after treatment, but be careful not to suck all the liquid out of the device.
      1. Add autoclaved ddH₂O to each well on either side of the device, and allow to flow to the other well by capillary action.
         
      2. Aspirate off the water (again being careful not to fully remove all the liquid from the device), then add another 150 μl of autoclaved ddH₂O to one of each connected well and allow it to flow through to the corresponding well.
         
      3. Repeat this quick wash twice, then place the device containing ddH₂O in an incubator at 37 °C for 1 h.
         
      4. Then, repeat the wash step again. Wash the devices at least three times with ddH₂O for one hour each, which is the total.
         
      5. Add NBM with the necessary factors (glutamax, B27, PenStrep) to the top wells. Let it flow through, incubate at 37 °C overnight.
         
      6. Prepare 40 ml plating media:
         800 μl B27 + 30 μg/ml gentamicin + 400 μl glutamax + 25 μM glutamate
         
      7. Prewarm 10 ml dissociation media in 15ml tube: Neurobasal + 5% FBS + gentamicin.
         
      8. Prewarm 10 ml plain neurobasal media for papain.
         
   Day 5:
   6. Add laminin (20 μg/ml) to each well and incubate at 37 °C for at least 30 min: Coat the chamber with laminin: take a 20 μl aliquot out from -80 °C freezer and put it on ice immediately, wait until it dissolves completely on ice then add cold neurobasal media to 1.8 ml.
      1. Turn the UV light on and UV sterilize one 6 cm cell culture dishs for 15 min.
         
      2. Pre-warm 14 ml plating media in this 6 cm cell culture dish, 37 °C incubator.
         
      3. Replace the laminin solution with plating media 1 h before plating the cells.
         
   
   
2. Dissection of hippocampal neurons
   1. Activate papain 10 mg aliquot with 750 μl papain buffer at 37 °C for 30 min.
      
   2. Add to prewarmed neurobasal media, filter sterialize, then add one DNase 20 μl aliquot.
      
   3. Wash the tissue with 10 ml cold dissection media in TC hood (wash twice).
      
   4. After the tissue settled by gravity, remove HBSS completely.
      
   5. Add one aliquot of DNase to 10 ml digestion media (prewarmed yesterday).
      
   6. Place half of digestion media (5 ml) onto the tissue for 15 min at 37 °C water bath.
      
   7. Add the remaining half of digestion media into the tube and incubate for another 15 min at 37 °C water bath.
      
   8. Let the tissue settle to bottom by gravity or spin shortly.
      
   9. Re-suspend the pellet with 5 ml papain dissociation media and triturate up and down with 5 ml pipette for 5 times, avoid bubbling the media.
      
   10. Let debris settle slowly, or short spin to 100 g up and down (600 rpm), transfer the supernatant to a new 15 ml tube.
       
   11. Continue triturate the undissociated tissues for 5-10 times, take the supernatant and combine with previous 5 ml.
       
   12. Take 10 μl out and mix with 10 μl trypan blue.
       
   13. Load 10 μl mixture on hemacytometer and count:
       All cells in 16 small squares x 10⁴ = cells/ml (A typical yield from 12 E18 pups is ~50 M in total).
       
   
   
3. Amaxa transfection
   1. Mix 82 μl nucleofector solution with 18 μl supplement before use.
      
   2. Prepare 50 μl nucleofector with 5 μg total DNA (if using more than 1 plasmid, mix in equimolar ratios).
      
   3. Spin down 5 million (1-6 million for each transfection) neurons at 150 x g 5 min. During spinning, transfer 7 ml pre-warmed plating media to a 15 ml tube, add 350 μl FBS, keep at 37 °C. Prepare the 1 ml pipette with a tip.
      
   4. After spinning, remove all media using aspiration pipette with a 200 μl pipette tip.
      
   5. Resuspend the cells in a 50 μl nucleofector solution.
      
   6. Mix 50 μl of the cells with the premix of DNA + neucleofector (a total of 100 μl).
      
   7. Zap with program G-13 (better for Hc) or O-03 (better for Cx). A foam of dead cells will form.
      
   8. Immediately pipette in 1 ml warmed plating medium with FBS and pipette out to a 1.7 ml eppendorf tube. Do not pick up dead foam. Be gentle.
      
   9. Incubate at 37 °C 10 min after rescue.
      
   10. Spin down the cells at 150 x g, 5 min.
       
   11. Resuspend in 300 μl plating media with FBS.
       
   12. Load 5 μl/well, filling the two upper wells of each microfluidic.
       
   13. Let cell adhere at 37 °C for 10 min.
       
   14. Fill the wells from both ends simultaneously with two pipettes with plating media with FBS.
       
   15. Completely change media to fresh plating media W/O FBS 2 h later.
       
   Day 6:
   16. Change media once again the next morning with plating media.
       
   17. Change half of the media every 2 days.
       

Recipes

1. 0.1 M borate buffer (pH 8.5) (borax boric acid buffer)
   1.24 g Boric acid
   1.9 g Borax
   Bring to 500 ml with ddH₂O
   PH should be 8.5 without adjustment
   Filter sterilize
   Store at 4 °C.
   
2. Dissection media (DM) (for 1L)
   82 ml 1 M Na₂SO₄
   60 ml 0.5 M K₂SO₄
   5.8 ml 1 M MgCl₂
   252 μl 1M CaCl₂
   8 ml 2.5 M glucose (20 mM)
   1 ml 1 M Hepes (pH 7.4)
   2 ml 0.5% Phenol Red pH with 1 M NaOH to 7.4 filter sterilize and keep at 4 °C.
   OR just use HBSS and add glucose to 20 mM, HEPES:
   500 ml HBSS with Ca²⁺ and Mg²⁺
   5 ml 1.5 M D-glucose (filter sterilized)
   5 ml 1.0 M HEPES pH 7.2
   
3. Papain activation buffer (50 ml)
   1.1 mM EDTA 110 μl (0.5 M EDTA, pH 8.0)
   0.067 mM β-mercaptoethanol 0.25 μl (0.0047 μl BME/ml buffer)
   5.5 mM cysteine 33.25 mg
   Store at 4 °C.
   
4. Papain
   Aliquot into 10 mg/1.7 ml eppendorf tube, Store lyophilized powder at 4 °C
   Add 750 μl papain buffer before use
   Activate to 200 U/ml (10 x) - 30 min at 37 °C (shake the tube once in a while)
   
5. DNase
   1,000 U/μl stock, 20 μl aliquot (20,000 U/tube, used in 10 ml)
   
6. Papain digestion media (for every ten animals)
   Add the 750 μl activated papain to plain neurobasal media
   Filter sterilize
   Add DNase: 2 U/μl DNase
   
7. Dissociation media (in 10 ml neurobasal)
   Gentamicin 40 μg/ml
   FBS 500 μl
   
8. Plating media with phenol red (in 20 ml neurobasal) with serum
   B27 400 μl (Gem21, NS21 all the same)
   Gentamicin 40 μg/ml (10 mg/ml stock, take 80 μl/20 ml media)
   Glutamax 200 μl
   Glutamate 25 μM (100 mM stock, 4,000x, 5 μl/20 ml) (use at 12.5 μM final concentration may be better: 2.5~3 μl/20 ml)
   FBS 200 μl
   
9. Growth media without phenol red (in 20 ml neurobasal)
   B27 400 μl (Gem21, NS21 all the same)
   Glutamax 200 μl
   

Acknowledgments

Alternative reference protocol can be found on the Millipore website related to AXIS^TM Axon Isolation Devices. The dissection protocol is modified from a protocol developed in Dr. Michael Lin’s lab, Department of Pediatrics and Bioengineering, Stanford University, USA.

References

1. Detailed reference protocols can be requested from http://www.xonamicrofluidics.com/about.html.
2. Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W. and Jeon, N. L. (2006). Microfluidic culture platform for neuroscience research. Nat Protoc 1(4): 2128-2136.