2.14. Isolation of Primary Liver Sinusoidal Endothelial Cells

Cell isolation from 3 Mice (SECs, KCs & HSCs)
 

Pronase E (Roche 10-165-921-001)
Collagenase D (Roche 11-088-874-103)
DNase I (Roche 10-104-159-001)
 

1)    Preparation of 6 kinds of solution
    A) SC-1 solution 200ml
    B) SC-2 solution 150ml + pronase E 50mg
    C) SC-2 solution 150ml + collagenase D 100mg
    D) SC-2 100ml + pronase E 50mg+collagenase D 50mg + DNase 1ml (DNase I solution: Adjust 2mg/ml using GBSS/B. Store at -20ºC)
    E) Nycodenz 1 (AXIS-SHIELD): 5.18g/total volime 15ml by GBSS/A  Filtered through 0.2um.
    F) Nycodenz 2 (AXIS-SHIELD): 3.63g/total volume 25ml by  GBSS/A  Filtered through 0.2um.

2)    Filter those solutions (0.2m Bottle top filter)
3)    Incubate A), B) and C) in the water bath (40ºC)
4)    Insert 27G catheter into Portal vein and fasten by a vascular crump and cut IVC.
5)    Perfuse (5ml/min) A) SC-1 solutionfor 5min.
6)    Perfuse (5ml/min) B) pronase E/SC-2 solution for 5 min.
7)    Perfuse (5ml/min) C) collagenase D/SC-2 solution for 5min.
8)    Take the liver out. Keep on ice in 50ml SC-2 solution.
9)    Tear membrane gently
10) Transfer the tissue into beaker. Add D) solution (collagenase 50mg + pronase 50mg + DNase 1ml/ total volume　150ml by SC-2 solution).
11) Incubate at 37ºC for 20 minutes with gently stirring. 
12) Filter the digested liver though a cell strainer (70μm nylon).
13) Transfer to 3 Falcon tube (50ml). Centrifuge 500 rpm for 1min at 4ºC.
14) Transfer supernatant to 3 Falcon tube (50ml). Discard or  keep the pellet (Hepatocyte).Centrifuge 2000 rpm for 8 min  at 4ºC. 
15) Resuspend each pellet in 10ml of GBSS/B containing 150μl of DNase I.
16) Collect each cell suspension into 2 Falcon tube (50ml).
17) Add GBSS/B up to 50ml. Centrifuge 2000 rpm (500g) for 8 min at 4ºC.
18) Discard supernatant.
19) Add 10ml of GBSS/B containing 150μl of DNase I andresuspend pellet.
20) Collect each cell suspension into 1 Falcon tube (50ml).
21) Add GBSS/B up to 34ml and add 14ml of E) Nycodenz 1 solution. Mix well. Total volume:50ml
22) Prepare 14.5% F) Nycodenz 2 solution (3.63g Nycodenz /GBSS-A 25ml). Transfer 4ml into 6 tubes (FALCON 15ml tube).
23) Lay 8.3ml of 21) onto the 14.5% Nycodenz solution.
24) Lay 1ml of GBSS/B onto the solution.
25) Centrifuge 3000rpm for 20min at 4ºC. Do not use Break!
26) Under the first layer of clear GBSS/B solution, you can see white layer. This layer contains stellate cell. Collect this layer and transfer to new 50ml tube. The second layer contains SEC & KC. Collect this layer and transfer to new 50ml tube.
27) Add GBSS/B solution to wash and centrifuge 1500rpm for 7min at 4ºC .
28) HSC; Count cells. Culture in DMEM+10% FBS on a plastic dish.
29) KC; Plate for 20min on a plastic dish and wash 3times by PBS. Culture in RPMI1640 +10%FBS. 
SEC; Supernatant contains SEC. If you need the high purity, you should do MACS procedure for SEC. SEC should be culture on the collagen coated plate in special medium.)
CC-3156 EBM-2 Basal Medium 500ml
CC-4176 EBM-2 Single Quot Kit Suppl. & Growth Factors.

(Clonetics, LONZA) 
                                    SC-1                  SC-2
NaCl                         8000mg/L            8000mg/L
KCl                            400mg/L              400mg/L
NaH₂PO₄. H₂O         88.17mg/L            88.17mg/L
Na₂HPO₄                  120.45mg/L           120.45mg/L
HEPES                       2380mg/L              2380mg/L
NaHCO₃                   350mg/L               350mg/L
EGTA                         190mg/L                 (-)
Glucose                     900mg/L                 (-)
CaCl₂. 2H₂O                 (-)                     560mg/L
                                pH: 7.20~7.30 by 1N NaOH

GBSS (Gey’s balanced salt solution)

                             GBSS-A          GBSS-B 
NaCl                      (-)                   8000mg/L
KCl                        370mg/L         370mg/L
MgCl₂. 6H₂O        210mg/L         210mg/L
MgSO₄. 7H₂O       70mg/L           70mg/L
Na₂HPO₄.              59.6mg/L        59.6mg/L
KH₂PO₄                  30mg/L          30mg/L
Glucose                  991mg/L         991mg/L
NaHCO₃                 227mg/L         227mg/L
CaCl₂. 2H₂O            225mg/L         225mg/L

                           pH: 7.20～7.30

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